Engineered phenylalanine ammonia lyase polypeptides

ABSTRACT

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of co-pending U.S. patentapplication Ser. No. 15/720,198, filed Sep. 29, 2017, which is aContinuation of co-pending U.S. patent application Ser. No. 15/431,491,filed Feb. 13, 2017, which is a Divisional of U.S. patent applicationSer. No. 14/255,539, filed Apr. 17, 2014, now U.S. Pat. No. 9,611,468,which claims priority to U.S. Prov. Pat. Appln. Ser. No. 61/813,586filed Apr. 18, 2013, and U.S. Prov. Pat. Appln. Ser. No. 61/897,932,filed Oct. 31, 2013, all of which are incorporated by reference in theirentireties for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file CX7-131US2_ST25.TXT, created onApr. 15, 2014, 127,412 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides engineered phenylalanine ammonia-lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.In some embodiments, the engineered PAL polypeptides are optimized toprovide enhanced catalytic activity, as well as reduced sensitivity toproteolysis and increased tolerance to acidic pH levels. In someembodiments the engineered PAL polypeptides are deimmunized. Theinvention also relates to the use of the compositions comprising theengineered PAL polypeptides for therapeutic and industrial purposes.

BACKGROUND OF THE INVENTION

Phenylalanine ammonia-lyase (PAL) along with histidine ammonia-lyase(HAL) and tyrosine ammonia-lyase (TAL) are members of the aromatic aminoacid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3). More specifically theenzymes having PAL activity (EC 4.3.1.23-1.25 and previously classifiedas EC4.3.1.5) catalyze the nonoxidative deamination of L-phenylalanineinto (E)-cinnamic acid. PAL is a non-mammalian enzyme that is widelydistributed in plants and has also been identified in fungi and alimited number of bacteria. PAL enzymes may be used as a therapeuticprotein for the treatment of the metabolic disorder, phenylketonuria(PKU). PKU is an autosomal metabolic genetic disorder in which thehepatic enzyme phenylalanine hydroxylase (PAH) or one or more of theenzymes involved in the synthesis or recycling of the co-factortetrahydrobiopterin, is nonfunctional due to a mutation in one of thecorresponding genes. This lack of functionality results in high levelsof phenylalanine in the bloodstream. The phenylalanine is converted intophenylpyruvate (phenylketone) and other derivatives. In humans, if PKUis not treated early, high levels of phenylalanine and some of itsbreakdown products can cause significant medical problems includingintellectual disability, microcephaly and seizures. Numerous studieshave focused on the use of PAL in the treatment of PKU by enzymesubstitution (Ambrus et al., Science 201:837-839 [1978]; Bourget et al.,Appl. Biochem. Biotechnol., 10:57-59 [1984]; and Sarkissian et al.,Proc. Natl. Acad. Sci. USA 96:2339-2344 [1999]).

One method of detoxifying phenylalanine in the blood stream is the useof injectable recombinant PAL and PAL variants modified by pegylation(PEG-PAL). Pegylation has been shown to improve enzyme half-life andreduce subject antigenic response (See e.g., WO 2008/153776, WO2011/097335, and U.S. Pat. No. 7,531,341). PAL variants useful inPEG-PAL compositions have been described as variants of wild-type Nostocpunctiforme (NpPAL); Anabaena variabilis (AvPAL) and Rhodosporidiumtoruloides (RtPAL). In particular, variants of wild-type AvPAL have beendescribed wherein the cysteine residues at positions 64, 318, 503 and565 have been substituted with serine (See e.g., U.S. Pat. Nos.7,790,433; 7,560,263; and 7,537,923).

An alternative route of PAL administration as a means of reducing plasmaconcentration of L-phenylalanine in PKU subjects is a non-invasiveformulation such as an oral formulation (Sarkissian et al., Proc. Natl.Acad. Sci. USA 96:2339-2344 [1999]). A key advantage of oral delivery ofPAL is the reduced exposure of the enzyme to the immune system therebyminimizing the immune response which is observed with injectablePEG-PAL. However, a major limitation for the oral formulation of PAL isloss of enzyme activity in the stomach and intestinal lumen. In order tobe effective and functional PAL must resist degradation by acidic pHsand proteases such as trypsin, chymotrypsin, carboxypeptidases andpepsin that normally degrade proteinaceous foods to oligopeptides andamino acids. In some previous studies (Sarkissian, supra) in order toachieve a significant effect for the oral administration of PAL, a largeamount of the enzyme was required partly due to enzymatic degradation byproteases and partly due to relatively low specific activity at pH 7.0.Various means have been explored to suppress PAL degradation upondigestion (Kim et al., Molec. Therap., 10:220-224 [2004]; and Shah etal., Int. J. Pharmaceut., 356:61-68 [2008]).

One approach to increase the effectiveness of PAL under the harshconditions of the digestive tract is to provide engineered PALpolypeptides that are tolerant to the inherent harsh conditions. Kang etal. used site directed mutagenesis of a chymotrypsin cleavage site andpegylation of surface lysines of an AvPAL to reduce proteolyticinactivation (See, Kang et al., Mol. Gen. Metabol., 99:4-9 [2010]). Inthese studies ten cleavage sites were specifically mutated and all buttwo of these resulting mutants (F18A and R94G) lost more than 50% of theoriginal enzyme activity. None of the mutants showed increased activityand the F18A mutant showed a slight increase in trypsin resistance (Kanget al., supra). Further studies with PAL, while effective, generallyhave not resulted in a longer lived enzyme. Therefore, oraladministration of previously described PAL mutants and derivativesthereof does not result in effective treatment of PKU.

Despite the progress made with various formulations of PAL there remainsa need for PAL polypeptides having improved properties for oraladministration. These improved properties include without limitation agreater half-life, increased catalytic activity, improved stability tothe conditions in the digestive track and reduced aggregation.

In addition to therapeutic applications PAL enzymes may also be used inthe industrial synthesis of L-phenylalanine and other substitutedL-phenylalanine derivatives. These derivatives may then be used aspharmaceutical precursors (Gloge et al., Chem., 6: 3386-3390 [2000];Bartsch et al., Prot. Eng. Des. Sel., 23:929-933 [2010]; and Turner,Curr. Opin. Chem. Biol., 234-240 [2011]).

PAL enzymes may also be used in agricultural applications. PAL plays asignificant role in biosynthesis of phenylpropanoids (such as flavonoidsand lignin) in plants, fungi and bacteria and can act as a defenserelated enzyme (Bate et al., Proc. Natl. Acad. Sci. USA 91:7608-7612[1994]). Modulation of PAL activity by using recombinant polypeptideshaving PAL activity could potentially lead to effective herbicides.

SUMMARY OF THE INVENTION

The present invention provides engineered phenylalanine ammonia-lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.In some embodiments, the engineered PAL polypeptides are optimized toprovide enhanced catalytic activity, as well as reduced sensitivity toproteolysis and increased tolerance to acidic pH levels. In someembodiments the engineered PAL polypeptides are deimmunized. Theinvention also relates to the use of the compositions comprising theengineered PAL polypeptides for therapeutic and industrial purposes. Insome embodiments, the present invention is directed to engineeredphenylalanine ammonia-lyase (PAL) polypeptides and biologically activefragments and analogs thereof having improved properties such anincreased tolerance to acidic pH and/or reduced sensitivity toproteolysis.

The present invention is directed to engineered PAL polypeptides andbiologically active fragments and analogs thereof having improvedproperties when compared to a wild-type PAL enzyme or a reference PALpolypeptide under essentially the same conditions. The invention isfurther directed to methods of using the engineered PAL polypeptides andbiologically active fragments and analogs thereof in therapeutic and/orindustrial compositions and methods of using such compositions fortherapeutic and/or industrial purposes.

In a first aspect, the invention provides engineered phenylalanineammonia-lyase (PAL) polypeptides wherein the engineered PAL polypeptidehas an improved property selected from the group of i) enhancedcatalytic activity, ii) reduced sensitivity to proteolysis, iii)increased tolerance to acidic pH, iv) reduced aggregation, or acombination of any of i), ii), iii) or iv) as compared to the referencesequence when measured under essentially the same conditions. In somespecific embodiments, the engineered PAL polypeptides have two improvedproperties. In other specific embodiments, the improved property isreduced sensitivity to proteolysis and in yet in other specificembodiments, the improved property is increased tolerance to acidic pH.

In a second aspect, the engineered PAL polypeptides include proteinscomprising at least 85% amino acid sequence identity to SEQ ID NO:4, ora functional fragment thereof and an amino acid residue difference at aposition corresponding to positions X39; X91; X158; X180; X195; X243;X245; X256; X257; X270; X290; X307; X308; X326; X349; X364; X394; X399;X400; X404; X407; X443; X453; X459; X460; X463; X474; X522; X524; andX528, when optimally aligned with the polypeptide of SEQ ID NO:4.

In some specific embodiments of the first and second aspects, theengineered PAL polypeptides comprise at least an amino acid residuedifference of one or more amino acid residue positions corresponding toA39; A91; Y158; S180; K195; T243; I245; A256; L257; N270; N290; H307;E308; I326; L349; L364; A394; S399; N400; P404; L407; F443; N453; Y459;T460; T463; N474; K522; T524; and P528, when optimally aligned with thepolypeptide of SEQ ID NO:4. In some specific embodiments, the engineeredPAL polypeptides comprise at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least15, and at least 20 amino acid residue differences from the referencepolypeptide comprising the amino acid sequence of SEQ ID NO:4.

In other specific embodiments of the first and second aspects, theengineered PAL polypeptides comprise at least 90%, (at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% and 99%) amino acid sequence identity toSEQ ID NO:4. In yet further specific embodiments, the engineered PALpolypeptides comprise at least 90%, (at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and 99%) amino acid sequence identity to SEQ ID NO:4, andcomprise one or more of the following substitutions A39V; A91V; Y158H;S180A; K195E; T243I/L; I245L; A256G; L257W/A; N270K; N290G; H307G/Q/M;E308Q; I326F; L349M; L364Q; A394V; S399N; N400K; P404A; L407V; F443H;N453G; Y459F; T460G; T463N; N474Q; K522Y/F/N; T524S; and P528L.

In other specific embodiments, the engineered PAL polypeptides arederived from a wild-type Anabaena variabilis PAL (such as ATCC29413;NCBI protein reference sequence YP_324488.1; SEQ ID NO:4).

In a third aspect, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity encompassed by the invention comprises anamino acid sequence having at least 99% sequence identity to SEQ IDNO:10, or a functional fragment thereof.

In a fourth aspect, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity encompassed by the invention comprise anamino acid sequence having at least 95% sequence identity to SEQ IDNO:10, or a functional fragment thereof and further comprising an aminoacid residue difference as compared to SEQ ID NO:10, at one, two, three,four, five, or six more amino acid positions.

In a fifth aspect, the invention provides a polynucleotide sequenceencoding any one of the engineered PAL polypeptides as described herein.

In a sixth aspect, the invention provides a pharmaceutical compositionor an industrial composition comprising any one of the engineered PALpolypeptides as described herein.

In some embodiments, the present invention provides engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activitycomprising a) an amino acid sequence having at least 85% sequenceidentity to reference sequence SEQ ID NO:4 or a functional fragmentthereof; b) an amino acid residue difference as compared to SEQ ID NO:4or the functional fragment thereof at one or more amino acid positions;and c) which exhibits an improved property selected from i) enhancedcatalytic activity, ii) reduced sensitivity to proteolysis, iii)increased tolerance to acidic pH, iv) reduced aggregation or acombination of any of i), ii), iii) or iv) as compared to the referencesequence. In some embodiments, one or more amino acid positions areselected from X39; X54; X59; X73; X91; X158; X112, X134, X180; X195;X240; X243; X245; X256; X257; X270; X290; X304, X305; X307; X308; X326;X349; X353; X364; X394; X399; X400; X404; X407; X443; X453; X459; X460;X463; X474; X509; X521; X522; X524; X528; X546; X564; and/orcombinations thereof when optimally aligned with the amino acid sequenceof SEQ ID NO: 4. In some additional embodiments, the improved propertyis selected from reduced sensitivity to proteolysis and/or increasedtolerance to acidic pH. In yet additional embodiments, the referencesequence is a wild-type PAL derived from Anabaena variabilis. In somefurther embodiments, the amino acid residue of the reference sequence ofSEQ ID NO:4 corresponds to A39; T54; G59, S73; A91; Y158; S180; K195;A112; R134; Q240; T243; I245; A256; L257; N270; N290; Y304; R305; H307;E308; I326; L349; D353; L364; A394; S399; N400; P404; L407; F443; N453;Y459; T460; T463; N474; E509; Q521; K522; T524; P528; S546; and/or P564.In some embodiments, the amino acid residue difference as compared toSEQ ID NO:4 is selected from one or more of the following substitutionsA39V; T54K; G59R; S73K; A112C; R134Q; A91V; Y158H; S180A; K195E;Q240R/W; T243I/L; I245L; A256G; L257W/A; N270K; N290G; Y304H; R305M;H307G/Q/M; E308Q; I326F; L349M; D353A/N; L364Q; A394V; S399N; N400K;P404A; L407V; F443H; N453G; Y459F; T460G; T463N; N474Q; E509L; Q521K/S;K522Y/F/N; T524S; P528L; S546R; and P564 G/L/M; when optimally alignedwith the polypeptide of SEQ ID NO:4. In some further embodiments, theengineered polypeptide has at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or about 100% sequence identity to reference sequenceSEQ ID NO:4. In some further embodiments, the engineered polypeptide hasat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to reference sequence SEQ ID NO:4. In someadditional embodiments, the engineered polypeptide has at least about90% sequence identity to reference sequence SEQ ID NO:4. In someadditional embodiments, the engineered polypeptide has at least about95% sequence identity to reference sequence SEQ ID NO:4. In some furtherembodiments, the engineered polypeptide has at least about 90% sequenceidentity to SEQ ID NO:4; and an amino acid residue difference atposition H307. In some further embodiments, the engineered polypeptidehas at least 90% sequence identity to reference sequence SEQ ID NO:4. Insome additional embodiments, the engineered polypeptide has at least 95%sequence identity to reference sequence SEQ ID NO:4. In some furtherembodiments, the engineered polypeptide has at least 90% sequenceidentity to SEQ ID NO:4; and an amino acid residue difference atposition H307. In some additional embodiments, the amino acid residuedifference is H307G/Q/M. In some further embodiments, the amino acidresidue difference is selected from a combination of one or more of A39;A91; Q240; A256; N290; Y304; R305; H307; D353 A394; S399; P404; L407;Q521; K522; and T524.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising an amino acidsequence having at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or at least about 100% sequence identity to SEQ ID NO:6, 8, 10, 12,and/or 14, or a functional fragment thereof. In some embodiments, theengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity comprise an amino acid sequence having at least about 95%sequence identity to SEQ ID NO:6, 8, 10, 12, and/or 14, or a functionalfragment thereof.

In some further embodiments, the engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprise an amino acidsequence having at least about 99% sequence identity to SEQ ID NO:6, 8,10, 12, and/or 14, or a functional fragment thereof.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising an amino acidsequence having at least about at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at 100% sequence identity to SEQ ID NO:6, 8,10, 12, and/or 14, or a functional fragment thereof. In someembodiments, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity comprise an amino acid sequence having atleast about 95% sequence identity to SEQ ID NO:6, 8, 10, 12, and/or 14,or a functional fragment thereof. In some further embodiments, theengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity comprise an amino acid sequence having at least about 99%sequence identity to SEQ ID NO:6, 8, 10, 12, and/or 14, or a functionalfragment thereof. In some embodiments, the engineered polypeptideshaving phenylalanine ammonia-lyase (PAL) activity comprise an amino acidsequence having at least 95% sequence identity to SEQ ID NO:6, 8, 10,12, and/or 14, or a functional fragment thereof. In some furtherembodiments, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity comprise an amino acid sequence having atleast 99% sequence identity to SEQ ID NO:6, 8, 10, 12, and/or 14, or afunctional fragment thereof.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising an amino acidsequence having at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or at least about 100% sequence identity to SEQ ID NO:4, or afunctional fragment thereof, wherein the engineered polypeptide isdeimmunized. The present invention also provides engineered polypeptideshaving phenylalanine ammonia-lyase (PAL) activity comprising an aminoacid sequence having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, or afunctional fragment thereof, wherein the engineered polypeptide isdeimmunized. In some embodiments, the engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprise an amino acidsequence having at least 95% sequence identity to SEQ ID NO:4, or afunctional fragment thereof, wherein the engineered polypeptide isdeimmunized. In some additional embodiments, the engineered polypeptidehaving phenylalanine ammonia-lyase (PAL) activity is a variant PALprovided in any of Tables 9-1 through 9-7. In some embodiments, thedeimmunized engineered having phenylalanine ammonia-lyase (PAL) activitycomprises an amino acid sequence having at least 95% sequence identityto SEQ ID NO:6, 8, 10, 12, and/or 14.

The present invention also provides polynucleotide sequences encodingthe engineered polypeptides having PAL activity provided herein. In someembodiments, the polynucleotide sequence is operably linked to a controlsequence. The present invention further provides vectors comprising atleast one polynucleotide sequence encoding at least on engineeredpolypeptide having PAL activity. The present invention also provideshost cells transformed with at least one polynucleotide sequenceencoding an engineered polypeptide having PAL activity, as providedherein.

The present invention further provides methods of producing anengineered PAL polypeptide in a host cell, comprising culturing a hostcell comprising at least one polynucleotide encoding at least oneengineered PAL polypeptide under suitable culture conditions. Thepresent invention further provides methods of producing an engineeredPAL polypeptide in a host cell, comprising culturing a host cellcomprising a polynucleotide encoding the engineered PAL polypeptideunder suitable culture conditions. In some embodiments, the methodsfurther comprise recovering the engineered PAL polypeptide from theculture and/or host cells.

The present invention also provides compositions comprising at least oneengineered polypeptide having PAL activity, as provided herein. In someembodiments, the composition is a pharmaceutical composition furthercomprising a pharmaceutically acceptable carrier. The present inventionfurther provides uses of these compositions.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising: a) an amino acidsequence having at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or greater sequence identity to areference sequence having phenylalanine ammonia-lyase (PAL) activity, ora functional fragment thereof; b) a polypeptide sequence comprising atleast one amino acid residue difference as compared to a referencesequence having phenylalanine ammonia-lyase (PAL) activity, or thefunctional fragment thereof at one or more amino acid positions; and c)which exhibits an improved property selected from i) enhanced catalyticactivity, ii) reduced sensitivity to proteolysis, iii) increasedtolerance to acidic pH, iv) reduced aggregation, v) reducedimmunogenicity, or a combination of any of i), ii), iii), iv), or v), ascompared to the reference sequence having phenylalanine ammonia-lyase(PAL) activity. In some embodiments, the reference sequence is aprokaryotic PAL, while in some other embodiments, the reference sequenceis a eukaryotic PAL. In some embodiments, the reference sequence is abacterial PAL (e.g., Anabaena variabilis PAL), while in some otherembodiments it is a human or other PAL. In some further embodiments, thereference sequence is a wild-type sequence (e.g., wild-type A.variabilis PAL), while in some alternative embodiments, the referencesequence is a variant enzyme (e.g., an engineered polypeptide having PALactivity).

In some embodiments, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity of the present invention comprise: a) anamino acid sequence having at least about 85%, at least about 86%, atleast about 87%, at least about 88%, at least about 89%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or greater sequenceidentity to reference sequence SEQ ID NO:4 or a functional fragmentthereof; b) a polypeptide sequence comprising at least one amino acidresidue difference as compared to SEQ ID NO:4, or the functionalfragment thereof at one or more amino acid positions; and c) whichexhibits an improved property selected from i) enhanced catalyticactivity, ii) reduced sensitivity to proteolysis, iii) increasedtolerance to acidic pH, iv) reduced aggregation, v) reducedimmunogenicity, or a combination of any of i), ii), iii), iv), or v), ascompared to the reference sequence SEQ ID NO:4.

In some additional embodiments, the engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprise: a) an amino acidsequence having at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater sequence identity to reference sequence SEQ IDNO:4 or a functional fragment thereof; b) a polypeptide sequencecomprising at least one amino acid residue difference as compared to SEQID NO:4 or the functional fragment thereof at one or more amino acidpositions; and c) which exhibits an improved property selected from i)enhanced catalytic activity, ii) reduced sensitivity to proteolysis,iii) increased tolerance to acidic pH, iv) reduced aggregation, v)reduced immunogenicity, or a combination of any of i), ii), iii), iv),or v), as compared to the reference sequence SEQ ID NO:4.

In some embodiments, the engineered polypeptides having phenylalanineammonia lyase activity (PAL) comprise at least one substitution(s) atone or more of the following amino acid positions: 20, 24, 27, 39, 43,45, 47, 54, 58, 59, 62, 70, 73, 80, 82, 91, 94, 98, 104, 105, 110, 112,115, 117, 118, 119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131,133, 134, 135, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 150,151, 153, 154, 156, 157, 158, 159, 172, 174, 175, 176, 177, 178, 180,187, 191, 195, 199, 205, 206, 210, 212, 213, 214, 232, 240, 243, 245,247, 248, 250, 256, 257, 266, 270, 275, 278, 279, 285, 286, 289, 290,292, 304, 305, 307, 308, 309, 319, 321, 326, 331, 332, 334, 349, 353,355, 364, 365, 369, 370, 371, 372, 374, 375, 377, 378, 379, 381, 382,383, 384, 385, 387, 389, 394, 396, 399, 400, 403, 404, 407, 417, 418,425, 431, 432, 433, 434, 435, 436, 437, 438, 439, 443, 446, 447, 453,456, 459, 460, 461, 463, 471, 472, 473, 474, 475, 476, 477, 478, 479,482, 483, 503, 507, 509, 521, 522, 524, 525, 528, 538, 546, 547, 551,558, 560, 564, 565, and/or any combinations thereof, wherein the aminoacid positions are numbered with reference to SEQ ID NO:4. In someembodiments, the amino acid residue of the reference sequence of SEQ IDNO:4 corresponds to A39, T54, G59, S73, A91, Y158, S180, K195, A112,R134, Q240, T243, I245, A256, L257, N270, N290, Y304, R305, H307, E308,I326, L349, D353, L364, A394, S399, N400, P404, L407, F443, N453, Y459,T460, T463, N474, E509, Q521, K522, T524, P528, S546, and/or P564. Insome additional embodiments, the amino acid residue difference ascompared to SEQ ID NO:4 is selected from one or more of the followingsubstitutions A39V, T54K, G59R, S73K, A112C, R134Q, A91V, Y158H, S180A,K195E, Q240R/W, T243I/L, I245L, A256G, L257W/A, N270K, N290G, Y304H,R305M, H307G/Q/M, E308Q, I326F, L349M, D353A/N, L364Q, A394V, S399N,N400K, P404A, L407V, F443H, N453G, Y459F, T460G, T463N, N474Q, E509L,Q521K/S, K522Y/F/N, T524S, P528L, S546R, and P564 G/L/M, when optimallyaligned with the polypeptide of SEQ ID NO:4. In some furtherembodiments, the engineered polypeptide has at least about 90% sequenceidentity to SEQ ID NO:4; and an amino acid residue difference atposition H307. In some embodiments, the amino acid residue difference isH307G/Q/M. In some still further embodiments, the amino acid residuedifference is selected from a combination of one or more of A39, A91,Q240, A256, N290, Y304, R305, H307, D353, A394, S399, P404, L407, Q521,K522, and T524. In some additional embodiments, the improved property ofthe engineered polypeptides having phenylalanine ammonia lyase activity(PAL) is selected from reduced sensitivity to proteolysis and/orincreased tolerance to acidic pH.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising: a) an amino acidsequence having at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or greater sequence identity toreference sequence SEQ ID NO:10 or a functional fragment thereof; b) apolypeptide sequence comprising at least one amino acid residuedifference as compared to SEQ ID NO:10 or the functional fragmentthereof at one or more amino acid positions; and c) which exhibits animproved property selected from i) enhanced catalytic activity, ii)reduced sensitivity to proteolysis, iii) increased tolerance to acidicpH, iv) reduced aggregation, v) reduced immunogenicity, or a combinationof any of i), ii), iii), iv), or v), as compared to the referencesequence SEQ ID NO:10.

In some embodiments, the present invention also provides engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activitycomprising: a) an amino acid sequence having at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater sequence identity toreference sequence SEQ ID NO:10 or a functional fragment thereof; b) apolypeptide sequence comprising at least one amino acid residuedifference as compared to SEQ ID NO:10 or the functional fragmentthereof at one or more amino acid positions; and c) which exhibits animproved property selected from i) enhanced catalytic activity, ii)reduced sensitivity to proteolysis, iii) increased tolerance to acidicpH, iv) reduced aggregation, v) reduced immunogenicity, or a combinationof any of i), ii), iii), iv), or v), as compared to the referencesequence SEQ ID NO:10.

In some embodiments, the present invention also provides engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activitycomprising: a) an amino acid sequence having at least 85% sequenceidentity to reference sequence SEQ ID NO:10 or a functional fragmentthereof; b) a polypeptide sequence comprising at least one amino acidresidue difference as compared to SEQ ID NO:10 or the functionalfragment thereof at one or more amino acid positions; and c) whichexhibits an improved property selected from i) enhanced catalyticactivity, ii) reduced sensitivity to proteolysis, iii) increasedtolerance to acidic pH, iv) reduced aggregation, v) reducedimmunogenicity, or a combination of any of i), ii), iii), iv), or v), ascompared to the reference sequence SEQ ID NO:10.

In some embodiments, the present invention also provide engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activitycomprising an amino acid sequence having at least 85% sequence identityto reference sequence SEQ ID NO:10 and at least one amino acid residuedifference as compared to SEQ ID NO:10 and that exhibits at least oneimproved property selected from enhanced catalytic activity, reducedsensitivity to proteolysis, increased tolerance to acidic pH, reducedaggregation, and/or reduced immunogenicity, as compared to the SEQ IDNO:10.

In some embodiments, the present invention also provides engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activity, whereinthe amino acid residue difference as compared to SEQ ID NO:10, isselected from one or more of the following substitutions or substitutionsets: I27E/V39A; I27E/V39A/R43L/V105C/A153R/L214E/P266H/L278D/C503Q;I27E/V39A/R43L/L214E/A547D;I27E/V39A/V105C/A112C/R134Q/L214E/L278D/C503Q/A547D/C565N;I27E/V39A/V105C/A112C/R134Q/A153R/Q205T/L214E/P266H/L278D/C503Q/A551D;I27E/V39A/V105C/A112C/Q205T/P210C/P266H/C503Q/A547D;I27E/V39A/V105C/A112C/Q205T/P266H/I285E/C503Q/A551D;I27E/V39A/V105C/A112C/L214E/I285E/C503Q/A547D;I27E/V39A/V105C/S131N/R134Q/Q205T/L214E/C503Q/A547D/C565N;I27E/V39A/V105C/R134Q/A153R/P210C/L278D/I285E/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/Q205T/P210C/L278D/C503Q/A547D;I27E/V39A/V105C/R134Q/Q205T/L214E;I27E/V39A/V105C/R134Q/Q205T/L214E/A551D/C565N;I27E/V39A/V105C/R134Q/Q205T/L278D/I285E/C503Q/A547D/A551D/C565N;I27E/V39A/V105C/R134Q/P210C; I27E/V39A/V105C/R134Q/P210C/L214E;I27E/V39A/V105C/R134Q/P210C/L214E/I285E/A547D;I27E/V39A/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N;I27E/V39A/V105C/R134Q/L214E/L278D/A547D/A551D;I27E/V39A/V105C/R134Q/L214E/I285E/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/P266H/C503Q;I27E/V39A/V105C/R134Q/P266H/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/L278D/C503Q/C565N;I27E/V39A/V105C/R134Q/L278D/I285E/C503Q;I27E/V39A/V105C/R134Q/L278D/A551D;I27E/V39A/V105C/R134Q/I285E/A547D/A551D;I27E/V39A/V105C/R134Q/C503Q/A551D;I27E/V39A/V105C/A153R/Q205T/L278D/C503Q/A547D/A551D;I27E/V39A/V105C/A153R/L214E; I27E/V39A/V105C/A153R/I285E;I27E/V39A/V105C/A153R/C503Q/A547D/C565N;I27E/V39A/V105C/A153R/A551D/C565N;I27E/V39A/V105C/Q205T/P210C/L214E/L278D/A547D;I27E/V39A/V105C/Q205T/P210C/L278D/C503Q;I27E/V39A/V105C/Q205T/P210C/L278D/A547D;I27E/V39A/V105C/Q205T/L214E/L278D/C503Q/A547D;I27E/V39A/V105C/Q205T/L278D/C503Q/A547D;I27E/V39A/V105C/P210C/I285E/C503Q/A547D/A551D/C565N;I27E/V39A/V105C/P210C/L214E/P266H/L278D;I27E/V39A/V105C/L214E/P266H/C503Q/A547D/C565N;I27E/V39A/V105C/L214E/L278D/L309P/C503Q/A547D/A551D;I27E/V39A/V105C/L278D/C503Q/A547D/C565N; I27E/V39A/V105C/I285E/A547D;I27E/V39A/V105C/C503Q/A551D; I27E/V39A/V105C/C503Q/A547D/A551D/C565N;I27E/V39A/A112C/R134Q/Q205T/P210C/L214E/A551D/C565N;I27E/V39A/A112C/R134Q/L214E/P266H/A551D;I27E/V39A/A112C/R134Q/L214E/C503Q/A547D;I27E/V39A/A112C/R134Q/P266H/I285E;I27E/V39A/A112C/Q205T/L214E/P266H/C503Q/A551D/C565N;I27E/V39A/A112C/Q205T/L278D/I285E; I27E/V39A/A112C/L214E;I27E/V39A/A112C/L214E/L278D/C503Q/A547D/A551D; I27E/V39A/A112C/I285E;I27E/V39A/A112C/A547D; I27E/V39A/R134Q;I27E/V39A/R134Q/A153R/Q205T/L214E/P266H/C503Q;I27E/V39A/R134Q/A153R/P210C/L214E/L278D/I285E/A547D/C565N;I27E/V39A/R134Q/A153R/L214E/P266H/L278D/C503Q/A547D/C565N;I27E/V39A/R134Q/A153G/L214E/P266H/I285E/C503Q/A551D/C565N;I27E/V39A/R134Q/A153R/L214E/C503Q/A547D; I27E/V39A/R134Q/A153R/L278D;I27E/V39A/R134Q/A153R/L278D/A547D/A551D; I27E/V39A/R134Q/A153R/A547D;I27E/V39A/R134Q/Q205T/L214E/P266H/I285E/C503Q/A551D/C565N;I27E/V39A/R134Q/Q205T/P266H/C503Q/A551D/C565N;I27E/V39A/R134Q/P210C/L214E/C503Q; I27E/V39A/R134Q/P210C/C503Q/A551D;I27E/V39A/R134Q/L214E/P266H/A551D;I27E/V39A/R134Q/L278D/I285E/C503Q/A547D/A551D;I27E/V39A/R134Q/L278D/C503Q/A547D; I27E/V39A/R134Q/C503Q/A547D;I27E/V39A/R134Q/A547D/C565N; I27E/V39A/Q205T/L214E/C503Q/C565N;I27E/V39A/Q205T/P266H/I285E/A547D/A551D/C565N;I27E/V39A/Q205T/P266H/A551D; I27E/V39A/Q205T/L278D/C503Q/A551D/C565N;I27E/V39A/Q205T/L278D/C503Q/C565N; I27E/V39A/Q205T/C503Q/A547D/C565N;I27E/V39A/P210C/T212S; I27E/V39A/P210C/L214E/L278D/C503Q/A551D;I27E/V39A/P210C/L214E/I285E/C503Q/A551D;I27E/V39A/P210C/P266H/I285E/C503Q/A547D;I27E/V39A/P210C/P266H/C503Q/A551D; I27E/V39A/L214E;I27E/V39A/L214E/P266H/L278D/C503Q/A547D/A551D/C565N;I27E/V39A/L214E/L278D/C503Q; I27E/V39A/L214E/L278D/C503Q/A547D/C565N;I27E/V39A/L214E/C503Q/A551D; I27E/V39A/P266H; I27E/V39A/P266H/L278D;I27E/V39A/L278D; I27E/V39A/L278D/A547D;I27E/V39A/L278D/I285E/C503Q/A547D; I27E/V39A/L278D/C503Q/C565N;I27E/V39A/C503Q; I27E/G45D/Q205T/P266H/C565N; I27E/V105C;I27E/V105C/R134Q/A153R/P210C/L214E/C503Q/A547D;I27E/V105C/R134Q/A153R/I285E/A547D; I27E/V105C/R134Q/A153R/C503Q;I27E/V105C/R134Q/Q205T/P210C/C503Q;I27E/V105C/R134Q/Q205T/L214E/P266H/L278D/C503Q/C565N;I27E/V105C/Q205T/P266H/C503Q;I27E/V105C/R134Q/P210C/L214E/P266H/L278D/A551D/C565N;I27E/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N;I27E/V105C/R134Q/P210C/P266H/L278D/I285E/C503Q/A551D/C565N;I27E/V105C/R134Q/L214E/L278D/C503Q/A547D;I27E/V105C/R134Q/L214E/L278D/C503Q/A547D/A551D/C565N; I27E/V105C/Q205T;I27E/V105C/Q205T/L214E/P266H; I27E/V105C/Q205T/L214E/P266H/A551D/C565N;I27E/V105C/Q205T/L214E/L278D/I285E/C503Q/A547D/A551D/C565N;I27E/V105C/Q205T/C503Q/A547D/A551D/C565N; I27E/V105C/L214E;I27E/V105C/L214E/P266H/C503Q; I27E/V105C/L214E/I285E/A551D/C565N;I27E/V105C/L214E/A547D/A551D/C565N; I27E/V105C/L214E/A551D/C565N;I27E/V105C/P266H; I27E/V105C/P266H/I285E/C503Q/A547D/C565N;I27E/V105C/L278D/A547D; I27E/V105C/I285E/C503Q/A547D/A551D/C565N;I27E/V105C/C503Q/A547D/C565N; I27E/V105C/C503Q/A547D/A551D/C565N;I27E/A112C/R134Q/A153R/L214E/P266H/C503Q;I27E/A112C/R134Q/L278D/I285E/C503Q/A551D/C565N;I27E/A112C/R134Q/Q205T/L278D/C503Q; I27E/A112C/R134Q/Q205T/I285E/C503Q;I27E/A112C/Q205T/P266H/L278D/I285E/C503Q;I27E/A112C/P210C/L214E/C503Q/A547D; I27E/R134Q;I27E/R134Q/A153R/I285E/C503Q/A547D; I27E/R134Q/Q205T/I285E/C503Q/A551D;I27E/R134Q/Q205T/P266H/L278D/A547D; I27E/R134Q/P210C;I27E/R134Q/L214E/C503Q; I27E/R134Q/L214E/C503Q/A547D;I27E/R134Q/L214E/C503Q/A547D/A551D; I27E/R134Q/L214E/C503Q/C565N;I27E/R134Q/L278D/I285E/A551D/C565N; I27E/R134Q/I285E/C503Q;I27E/A153R/L214E/L278D/I285E/A551D/C565N; I27E/A153R/L214E/L278D/A551D;I27E/Q205T; I27E/Q205T/L214E/L278D/I285E/C503Q/C565N;I27E/Q205T/L214E/C503Q/A547D/C565N;I27E/Q205T/P266H/L278D/I285E/A551D/C565N; I27E/Q205T/L278D/A551D;I27E/P210C; I27E/P210C/L214E/C503Q/A547D; I27E/P210C/L278D/C503Q;I27E/P210C/C503Q; I27E/P210C/C503Q/C565N; I27E/P210C/A551D; I27E/L214E;I27E/L214E/P266H/L278D/I285E/A551D; I27E/L214E/L278D;I27E/L214E/L278D/C503Q; I27E/L214E/C503Q; I27E/L214E/C503Q/A547D;I27E/L214E/C503Q/A547D/C565N; I27E/L214E/A551D; I27E/P266H/L278D/C503Q;I27E/P266H/A547D/A551D; I27E/L278D/C503Q/A551D;I27E/L278D/C503Q/A551D/C565N; I27E/A547D/C565N;V39A/G45S/L278D/C503Q/A551D; V39A/V105C/R134Q/A153R/Q205T/A551D;V39A/V105C/R134Q/P210C/L214E/A551D;V39A/V105C/R134Q/L214E/C503Q/A547D/A551D;V39A/V105C/A153R/P266H/A547D/A551D; V39A/V105C/Q205T/C503Q;V39A/V105C/Q205T/A551D; V39A/V105C/P210C/A547D;V39A/V105C/L214E/P266H/A547D/C565N;V39A/V105C/L214E/I285E/C503Q/A551D/C565N;V39A/A112C/R134Q/Q205T/L214E/L278D;V39A/A112C/R134Q/L214E/C503Q/A547D/A551D;V39A/A112C/A153R/Q205T/L278D/C503Q/A547D; V39A/R134Q;V39A/R134Q/Q205T/L214E/C503Q/C565N; V39A/R134Q/P210C/L214E/A547D/C565N;V39A/A153R/C503Q/A547D; V39A/Q205T/L278D/A547D/A551D;V39A/P210C/L214E/L278D/I285E/C503Q/A551D; V39A/P266H;V39A/P275R/L278D/C503Q/A551D; V39A/C503Q; V39A/C503Q/A551D/C565N; V105C;V105C/A112C/R134Q/Q205T/L214E/Y492H/C503Q/A547D;V105C/R134Q/A153R/Q205T/L214E/C503Q; V105C/R134Q/Q205T/L214E/A547D;V105C/R134Q/Q205T/P266H/L278D;V105C/R134Q/L214E/P266H/I285E/C503Q/A551D/C565N;V105C/R134Q/L214E/L278D/C565N; V105C/R134Q/L214E/C503Q/A547D;V105C/R134Q/L214E/C503Q/A547D/A551D; V105C/R134Q/C503Q;V105C/R134Q/C503Q/A547D; V105C/R134Q/C503Q/A547D/C565N;V105C/A153R/Q205T/L214E/P266H/C503Q/A547D;V105C/A153R/Q205T/P266H/I285E/A547D/C565N;V105C/Q205T/P210C/L214E/C503Q/A547D; V105C/Q205T/L214E/L278D;V105C/Q205T/L214E/C503Q/A547D/A551D/C565N; V105C/Q205T/C503Q/A551D;V105C/L214E/P266H/L278D/A547D; V105C/L214E/L278D/C503Q/A547D/A551D;V105C/L214E/I285E; V105C/L214E/I285E/C503Q/A547D/A551D/C565N;V105C/L214E/I285E/A547D/C565N; V105C/L278D/C503Q/A551D; V105C/I285E;V105C/I285E/A547D; V105C/C503Q; V105C/A547D/A551D;A112C/R134Q/A153R/L214E/L278D/I285E/C503Q/A547D/A551D/C565N;A112C/R134Q/L214E/C503Q/A547D/A551D/C565N; A112C/L214E/L278D;A112C/L278D/C503Q/A547D; R134Q/Q205T/L214E/I285E/C503Q/A551D/C565N;R134Q/Q205T/C503Q; R134Q/P210C/L214E/L278D/C503Q/A547D/C565N;R134Q/P210C/L214E/C503Q/A547D/A551D; R134Q/L214E;R134Q/L214E/L278D/C503Q; R134Q/L214E/L278D/C503Q/A551D;R134Q/L214E/I285E/C503Q; R134Q/C503Q; R134Q/C503Q/A547D/A551D; A153R;Q205T/L214E/I285E/C503Q/A551D; Q205T/L214E/I285E/C503Q/C565N;Q205T/L214E/C503Q/A547D/C565N; Q205T/L278D/I285E/A547D/A551D;P210C/L214E; P210C/L214E/P266H; L214E/P266H;L214E/P266H/C503Q/A547D/A551D/C565N; L214E/C503Q/A547D; L214E/A547D;P266H/L278D/C503Q; P266H/C565N; L278D/A547D; C503Q; C503Q/A547D;C503Q/A547D/A551D/C565N; C503Q/A547D/C565N; C503Q/A551D;C503Q/A551D/C565N; A547D; and/or C565N.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: V80I/R134C/P564Q; V121C; A123G; A124G; M125L;L1261/T; L126M/R134L; L127A; A129G/L; N130Q; N130C/M370I R134W; M133R;R1341; R134N/G307C; G135C/S; and/or G135A/A394E.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: G20S/I144L; R43S; L47M/I144L; L47M/R146E;L47M/M147G/A383E; L47M/P157C; Q58H/L143V; Q58K/P157D/G369C; A62S/M147V;S82I/G135C/P157F/W279L; R94C/I149E; T110I/I139R; L118M/L141H;A119E/T156H/A289D; I139M/V; R140D/G/M; R140N/A199E; R140E/A334S/A551D;L141K/Q/P/T; E142H/P/V; E142D/G371D; L143F/M; I144L/N/V; K145N/Q/R;K145G/P157T; R146H/L; R146W/D191Y; M147A; I149L/R; F150K/L/M; L151M;A153C/G; A153S/H250N; G154R; G154Y/L174M/Q321K/S456I/G483C; T156K/G483C;P157D/F/H/Y; Y158E; V159C/H/L/M; M247I; L319M; and/or Q389K.

In still some additional embodiments, the present invention alsoprovides engineered polypeptides having phenylalanine ammonia-lyase(PAL) activity, wherein the amino acid residue difference as compared toSEQ ID NO:10, is selected from one or more of the followingsubstitutions or substitution sets: P117T/Y176Q; V172I/C/L; L174M;S175G; Y176E/I/M/R/V; I177M/V; T178L/A477S; and/or S180C/T.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: R43S/H374K; R43S/H374R; A112S/M370A/A507E;M147I/H374S; S187R/L381V; D191Y/H385N; A232S; Q240K/H374R; A256S/L381N;P275Q/M370S; P275T/H374R; Q332K/Y377M; A334S/H374V; L349M; Q355K/H374S;M370G/I/S; G371H/N/Q/S; M372A/V; H374A/D/G/L/N/R/S/T; H374Q/P396Q;H374R/G417C; L375I; L375M; Y377C/I/N; Y378C/D/E/I/L/N/S; Y378F/P404Q;I379C/H/L/M/N; L381G/V; L381M/Q560K; L382C/H/I/M/S; A383S/V; K384R;H385C/G/N; H385M/P403H; H385S/P403H; D387S; L418M; G425V; A447S; S461G;and/or S525L.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: A24S/F434M; A62S/T433N; S98I; L213M/S438L;Q240K/T433Y; S286R/Y435T; A289S/L431E; S331I; L431C/E/G/P/S/V; L432C/V;T433A/I/L/N/P/Q/R/S/V/W; F434C; Y435L; Y435Q/H446N; G436M; G436D/T;N437E/G/Q; N437T/L538M; S438C/F/M/R/T; I439C/F/L/V; and/or A477S.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: A24E; Q58R/Y475H; A70S/N474E; L104M/V476L;A119E/G365A; L206M; P275Q; G276V; Q292H/A479G; Q355H/I478C; P404T/A477V;I471F/G/K/M/N/R/V/W; F472G; Q473H/K/M/R/S; Q473H/A507S; N474A/H/R/W;N474D/R490H; Y475C/F/L/Q; V476C/I/L; I478N/S; A479G/S; F482C/L;G483C/H/S; G483A/S524I; G483R/G537C; and/or A558S.

In some additional embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: V39A/K115E/M133R/C565N; V39A/M133R/F472G/C503Q/C565N;V39A/M133R/F472G/C565N; V39A/M133R/C503Q; V39A/M133R/C503Q/C565N;V39A/M147A/Y378E/C503Q/C565N; V39A/M147A/Y378E/C565N;V39A/M147A/L381G/F472G/C503Q/C565N; V39A/M147A/L381G/C503Q/C565N;V39A/M147A/F472G/C503Q/C565N; V39A/M147A/F472G/C565N; V39A/M147A/C565N;V39A/G248C/L381G/F472G/C503Q/C565N; V39A/Y378E/C503Q/C565N;V39A/Y378E/C565N; V39A/L381G; V39A/F472G/C503Q/C565N; V39A/C503Q/C565N;M133R/L381G/C565N; M133R/C503Q; Y378D/C503Q; Y378E/F472G/C503Q/C565N;L381G/F472G/C503Q/C565N; and/or F472G/C503Q/C565N.

In still some further embodiments, the present invention also providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity, wherein the amino acid residue difference as compared to SEQID NO:10, is selected from one or more of the following substitutions orsubstitution sets: I27E/V39A;I27E/V39A/R43L/V105C/A153R/L214E/P266H/L278D/C503Q;I27E/V39A/R43L/L214E/A547D;I27E/V39A/V105C/A112C/R134Q/L214E/L278D/C503Q/A547D/C565N;I27E/V39A/V105C/A112C/R134Q/A153R/Q205T/L214E/P266H/L278D/C503Q/A551D;I27E/V39A/V105C/A112C/Q205T/P210C/P266H/C503Q/A547D;I27E/V39A/V105C/A112C/Q205T/P266H/I285E/C503Q/A551D;I27E/V39A/V105C/A112C/L214E/I285E/C503Q/A547D;I27E/V39A/V105C/5131N/R134Q/Q205T/L214E/C503Q/A547D/C565N;I27E/V39A/V105C/R134Q/A153R/P210C/L278D/I285E/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/Q205T/P210C/L278D/C503Q/A547D;I27E/V39A/V105C/R134Q/Q205T/L214E;I27E/V39A/V105C/R134Q/Q205T/L214E/A551D/C565N;I27E/V39A/V105C/R134Q/Q205T/L278D/I285E/C503Q/A547D/A551D/C565N;I27E/V39A/V105C/R134Q/P210C; I27E/V39A/V105C/R134Q/P210C/L214E;I27E/V39A/V105C/R134Q/P210C/L214E/I285E/A547D;I27E/V39A/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N;I27E/V39A/V105C/R134Q/L214E/L278D/A547D/A551D;I27E/V39A/V105C/R134Q/L214E/I285E/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/P266H/C503Q;I27E/V39A/V105C/R134Q/P266H/C503Q/A547D/A551D;I27E/V39A/V105C/R134Q/L278D/C503Q/C565N;I27E/V39A/V105C/R134Q/L278D/I285E/C503Q;I27E/V39A/V105C/R134Q/L278D/A551D;I27E/V39A/V105C/R134Q/I285E/A547D/A551D;I27E/V39A/V105C/R134Q/C503Q/A551D;I27E/V39A/V105C/A153R/Q205T/L278D/C503Q/A547D/A551D;I27E/V39A/V105C/A153R/L214E; I27E/V39A/V105C/A153R/I285E;I27E/V39A/V105C/A153R/C503Q/A547D/C565N;I27E/V39A/V105C/A153R/A551D/C565N;I27E/V39A/V105C/Q205T/P210C/L214E/L278D/A547D;I27E/V39A/V105C/Q205T/P210C/L278D/C503Q;I27E/V39A/V105C/Q205T/P210C/L278D/A547D;I27E/V39A/V105C/Q205T/L214E/L278D/C503Q/A547D;I27E/V39A/V105C/Q205T/L278D/C503Q/A547D;I27E/V39A/V105C/P210C/I285E/C503Q/A547D/A551D/C565N;I27E/V39A/V105C/P210C/L214E/P266H/L278D;I27E/V39A/V105C/L214E/P266H/C503Q/A547D/C565N;I27E/V39A/V105C/L214E/L278D/L309P/C503Q/A547D/A551D;I27E/V39A/V105C/L278D/C503Q/A547D/C565N; I27E/V39A/V105C/I285E/A547D;I27E/V39A/V105C/C503Q/A551D; I27E/V39A/V105C/C503Q/A547D/A551D/C565N;I27E/V39A/A112C/R134Q/Q205T/P210C/L214E/A551D/C565N;I27E/V39A/A112C/R134Q/L214E/P266H/A551D;I27E/V39A/A112C/R134Q/L214E/C503Q/A547D;I27E/V39A/A112C/R134Q/P266H/I285E;I27E/V39A/A112C/Q205T/L214E/P266H/C503Q/A551D/C565N;I27E/V39A/A112C/Q205T/L278D/I285E; I27E/V39A/A112C/L214E;I27E/V39A/A112C/L214E/L278D/C503Q/A547D/A551D; I27E/V39A/A112C/I285E;I27E/V39A/A112C/A547D; I27E/V39A/R134Q;I27E/V39A/R134Q/A153R/Q205T/L214E/P266H/C503Q;I27E/V39A/R134Q/A153R/P210C/L214E/L278D/I285E/A547D/C565N;I27E/V39A/R134Q/A153R/L214E/P266H/L278D/C503Q/A547D/C565N;I27E/V39A/R134Q/A153G/L214E/P266H/I285E/C503Q/A551D/C565N;I27E/V39A/R134Q/A153R/L214E/C503Q/A547D; I27E/V39A/R134Q/A153R/L278D;I27E/V39A/R134Q/A153R/L278D/A547D/A551D; I27E/V39A/R134Q/A153R/A547D;I27E/V39A/R134Q/Q205T/L214E/P266H/I285E/C503Q/A551D/C565N;I27E/V39A/R134Q/Q205T/P266H/C503Q/A551D/C565N;I27E/V39A/R134Q/P210C/L214E/C503Q; I27E/V39A/R134Q/P210C/C503Q/A551D;I27E/V39A/R134Q/L214E/P266H/A551D;I27E/V39A/R134Q/L278D/I285E/C503Q/A547D/A551D;I27E/V39A/R134Q/L278D/C503Q/A547D; I27E/V39A/R134Q/C503Q/A547D;I27E/V39A/R134Q/A547D/C565N; I27E/V39A/Q205T/L214E/C503Q/C565N;I27E/V39A/Q205T/P266H/I285E/A547D/A551D/C565N;I27E/V39A/Q205T/P266H/A551D; I27E/V39A/Q205T/L278D/C503Q/A551D/C565N;I27E/V39A/Q205T/L278D/C503Q/C565N; I27E/V39A/Q205T/C503Q/A547D/C565N;I27E/V39A/P210C/T212S; I27E/V39A/P210C/L214E/L278D/C503Q/A551D;I27E/V39A/P210C/L214E/I285E/C503Q/A551D;I27E/V39A/P210C/P266H/I285E/C503Q/A547D;I27E/V39A/P210C/P266H/C503Q/A551D; I27E/V39A/L214E;I27E/V39A/L214E/P266H/L278D/C503Q/A547D/A551D/C565N;I27E/V39A/L214E/L278D/C503Q; I27E/V39A/L214E/L278D/C503Q/A547D/C565N;I27E/V39A/L214E/C503Q/A551D; I27E/V39A/P266H; I27E/V39A/P266H/L278D;I27E/V39A/L278D; I27E/V39A/L278D/A547D;I27E/V39A/L278D/I285E/C503Q/A547D; I27E/V39A/L278D/C503Q/C565N;I27E/V39A/C503Q; I27E/G45D/Q205T/P266H/C565N; I27E/V105C;I27E/V105C/R134Q/A153R/P210C/L214E/C503Q/A547D;I27E/V105C/R134Q/A153R/I285E/A547D; I27E/V105C/R134Q/A153R/C503Q;I27E/V105C/R134Q/Q205T/P210C/C503Q;I27E/V105C/R134Q/Q205T/L214E/P266H/L278D/C503Q/C565N;I27E/V105C/Q205T/P266H/C503Q;I27E/V105C/R134Q/P210C/L214E/P266H/L278D/A551D/C565N;I27E/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N;I27E/V105C/R134Q/P210C/P266H/L278D/I285E/C503Q/A551D/C565N;I27E/V105C/R134Q/L214E/L278D/C503Q/A547D;I27E/V105C/R134Q/L214E/L278D/C503Q/A547D/A551D/C565N; I27E/V105C/Q205T;I27E/V105C/Q205T/L214E/P266H; I27E/V105C/Q205T/L214E/P266H/A551D/C565N;I27E/V105C/Q205T/L214E/L278D/I285E/C503Q/A547D/A551D/C565N;I27E/V105C/Q205T/C503Q/A547D/A551D/C565N; I27E/V105C/L214E;I27E/V105C/L214E/P266H/C503Q; I27E/V105C/L214E/I285E/A551D/C565N;I27E/V105C/L214E/A547D/A551D/C565N; I27E/V105C/L214E/A551D/C565N;I27E/V105C/P266H; I27E/V105C/P266H/I285E/C503Q/A547D/C565N;I27E/V105C/L278D/A547D; I27E/V105C/I285E/C503Q/A547D/A551D/C565N;I27E/V105C/C503Q/A547D/C565N; I27E/V105C/C503Q/A547D/A551D/C565N;I27E/A112C/R134Q/A153R/L214E/P266H/C503Q;I27E/A112C/R134Q/L278D/I285E/C503Q/A551D/C565N;I27E/A112C/R134Q/Q205T/L278D/C503Q; I27E/A112C/R134Q/Q205T/I285E/C503Q;I27E/A112C/Q205T/P266H/L278D/I285E/C503Q;I27E/A112C/P210C/L214E/C503Q/A547D; I27E/R134Q;I27E/R134Q/A153R/I285E/C503Q/A547D; I27E/R134Q/Q205T/I285E/C503Q/A551D;I27E/R134Q/Q205T/P266H/L278D/A547D; I27E/R134Q/P210C;I27E/R134Q/L214E/C503Q; I27E/R134Q/L214E/C503Q/A547D;I27E/R134Q/L214E/C503Q/A547D/A551D; I27E/R134Q/L214E/C503Q/C565N;I27E/R134Q/L278D/I285E/A551D/C565N; I27E/R134Q/I285E/C503Q;I27E/A153R/L214E/L278D/I285E/A551D/C565N; I27E/A153R/L214E/L278D/A551D;I27E/Q205T; I27E/Q205T/L214E/L278D/I285E/C503Q/C565N;I27E/Q205T/L214E/C503Q/A547D/C565N;I27E/Q205T/P266H/L278D/I285E/A551D/C565N; I27E/Q205T/L278D/A551D;I27E/P210C; I27E/P210C/L214E/C503Q/A547D; I27E/P210C/L278D/C503Q;I27E/P210C/C503Q; I27E/P210C/C503Q/C565N; I27E/P210C/A551D; I27E/L214E;I27E/L214E/P266H/L278D/I285E/A551D; I27E/L214E/L278D;I27E/L214E/L278D/C503Q; I27E/L214E/C503Q; I27E/L214E/C503Q/A547D;I27E/L214E/C503Q/A547D/C565N; I27E/L214E/A551D; I27E/P266H/L278D/C503Q;I27E/P266H/A547D/A551D; I27E/L278D/C503Q/A551D;I27E/L278D/C503Q/A551D/C565N; I27E/A547D/C565N;V39A/G45S/L278D/C503Q/A551D; V39A/V105C/R134Q/A153R/Q205T/A551D;V39A/V105C/R134Q/P210C/L214E/A551D;V39A/V105C/R134Q/L214E/C503Q/A547D/A551D;V39A/V105C/A153R/P266H/A547D/A551D; V39A/V105C/Q205T/C503Q;V39A/V105C/Q205T/A551D; V39A/V105C/P210C/A547D;V39A/V105C/L214E/P266H/A547D/C565N;V39A/V105C/L214E/I285E/C503Q/A551D/C565N;V39A/A112C/R134Q/Q205T/L214E/L278D;V39A/A112C/R134Q/L214E/C503Q/A547D/A551D;V39A/A112C/A153R/Q205T/L278D/C503Q/A547D; V39A/R134Q;V39A/R134Q/Q205T/L214E/C503Q/C565N; V39A/R134Q/P210C/L214E/A547D/C565N;V39A/A153R/C503Q/A547D; V39A/Q205T/L278D/A547D/A551D;V39A/P210C/L214E/L278D/I285E/C503Q/A551D; V39A/P266H;V39A/P275R/L278D/C503Q/A551D; V39A/C503Q; V39A/C503Q/A551D/C565N; V105C;V105C/A112C/R134Q/Q205T/L214E/Y492H/C503Q/A547D;V105C/R134Q/A153R/Q205T/L214E/C503Q; V105C/R134Q/Q205T/L214E/A547D;V105C/R134Q/Q205T/P266H/L278D;V105C/R134Q/L214E/P266H/I285E/C503Q/A551D/C565N;V105C/R134Q/L214E/L278D/C565N; V105C/R134Q/L214E/C503Q/A547D;V105C/R134Q/L214E/C503Q/A547D/A551D; V105C/R134Q/C503Q;V105C/R134Q/C503Q/A547D; V105C/R134Q/C503Q/A547D/C565N;V105C/A153R/Q205T/L214E/P266H/C503Q/A547D;V105C/A153R/Q205T/P266H/I285E/A547D/C565N;V105C/Q205T/P210C/L214E/C503Q/A547D; V105C/Q205T/L214E/L278D;V105C/Q205T/L214E/C503Q/A547D/A551D/C565N; V105C/Q205T/C503Q/A551D;V105C/L214E/P266H/L278D/A547D; V105C/L214E/L278D/C503Q/A547D/A551D;V105C/L214E/I285E; V105C/L214E/I285E/C503Q/A547D/A551D/C565N;V105C/L214E/I285E/A547D/C565N; V105C/L278D/C503Q/A551D; V105C/I285E;V105C/I285E/A547D; V105C/C503Q; V105C/A547D/A551D;A112C/R134Q/A153R/L214E/L278D/I285E/C503Q/A547D/A551D/C565N;A112C/R134Q/L214E/C503Q/A547D/A551D/C565N; A112C/L214E/L278D;A112C/L278D/C503Q/A547D; R134Q/Q205T/L214E/I285E/C503Q/A551D/C565N;R134Q/Q205T/C503Q; R134Q/P210C/L214E/L278D/C503Q/A547D/C565N;R134Q/P210C/L214E/C503Q/A547D/A551D; R134Q/L214E;R134Q/L214E/L278D/C503Q; R134Q/L214E/L278D/C503Q/A551D;R134Q/L214E/I285E/C503Q; R134Q/C503Q; R134Q/C503Q/A547D/A551D; A153R;Q205T/L214E/I285E/C503Q/A551D; Q205T/L214E/I285E/C503Q/C565N;Q205T/L214E/C503Q/A547D/C565N; Q205T/L278D/I285E/A547D/A551D;P210C/L214E; P210C/L214E/P266H; L214E/P266H;L214E/P266H/C503Q/A547D/A551D/C565N; L214E/C503Q/A547D; L214E/A547D;P266H/L278D/C503Q; P266H/C565N; L278D/A547D; C503Q; C503Q/A547D;C503Q/A547D/A551D/C565N; C503Q/A547D/C565N; C503Q/A551D;C503Q/A551D/C565N; A547D; C565N; V80I/R134C/P564Q; V121C; A123G; A124G;M125L; L1261/T; L126M/R134L; L127A; A129G/L; N130Q; N130C/M370I R134W;M133R; R1341; R134N/G307C; G135C/S; G135A/A394E; G20S/I144L; R43S;L47M/I144L; L47M/R146E; L47M/M147G/A383E; L47M/P157C; Q58H/L143V;Q58K/P157D/G369C; A62S/M147V; S82I/G135C/P157F/W279L; R94C/I149E;T110I/I139R; L118M/L141H; A119E/T156H/A289D; I139M/V; R140D/G/M;R140N/A199E; R140E/A334S/A551D; L141K/Q/P/T; E142H/P/V; E142D/G371D;L143F/M; I144L/N/V; K145N/Q/R; K145G/P157T; R146H/L; R146W/D191Y; M147A;I149L/R; F150K/L/M; L151M; A153C/G; A153S/H250N; G154R;G154Y/L174M/Q321K/S456I/G483C; T156K/G483C; P157D/F/H/Y; Y158E;V159C/H/L/M; M247I; L319M; Q389K; P117T/Y176Q; V172I/C/L; L174M; S175G;Y176E/I/M/R/V; I177M/V; T178L/A477S; S180C/T; R43S/H374K; R43S/H374R;A112S/M370A/A507E; M147I/H374S; S187R/L381V; D191Y/H385N; A232S;Q240K/H374R; A256S/L381N; P275Q/M370S; P275T/H374R; Q332K/Y377M;A334S/H374V; L349M; Q355K/H374S; M370G/I/S; G371H/N/Q/S; M372A/V;H374A/D/G/L/N/R/S/T; H374Q/P396Q; H374R/G417C; L375I; L375M; Y377C/I/N;Y378C/D/E/I/L/N/S; Y378F/P404Q; I379C/H/L/M/N; L381G/V; L381M/Q560K;L382C/H/I/M/S; A383S/V; K384R; H385C/G/N; H385M/P403H; H385S/P403H;D387S; L418M; G425V; A447S; S461G; S525L; A24S/F434M; A62S/T433N; S98I;L213M/S438L; Q240K/T433Y; S286R/Y435T; A289S/L431E; S331I;L431C/E/G/P/S/V; L432C/V; T433A/I/L/N/P/Q/R/S/V/W; F434C; Y435L;Y435Q/H446N; G436M; G436D/T; N437E/G/Q; N437T/L538M; S438C/F/M/R/T;I439C/F/L/V; A477S; A24E; Q58R/Y475H; A70S/N474E; L104M/V476L;A119E/G365A; L206M; P275Q; G276V; Q292H/A479G; Q355H/I478C; P404T/A477V;I471F/G/K/M/N/R/V/W; F472G; Q473H/K/M/R/S; Q473H/A507S; N474A/H/R/W;N474D/R490H; Y475C/F/L/Q; V476C/I/L; I478N/S; A479G/S; F482C/L;G483C/H/S; G483A/S524I; G483R/G537C; A558S; V39A/K115E/M133R/C565N;V39A/M133R/F472G/C503Q/C565N; V39A/M133R/F472G/C565N; V39A/M133R/C503Q;V39A/M133R/C503Q/C565N; V39A/M147A/Y378E/C503Q/C565N;V39A/M147A/Y378E/C565N; V39A/M147A/L381G/F472G/C503Q/C565N;V39A/M147A/L381G/C503Q/C565N; V39A/M147A/F472G/C503Q/C565N;V39A/M147A/F472G/C565N; V39A/M147A/C565N;V39A/G248C/L381G/F472G/C503Q/C565N; V39A/Y378E/C503Q/C565N;V39A/Y378E/C565N; V39A/L381G; V39A/F472G/C503Q/C565N; V39A/C503Q/C565N;M133R/L381G/C565N; M133R/C503Q; Y378D/C503Q; Y378E/F472G/C503Q/C565N;L381G/F472GC503Q/C565N; and/or F472G/C503Q/C565N.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity comprising: a) an amino acidsequence having at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or greater sequence identity toreference sequence SEQ ID NO:26 or a functional fragment thereof; b) apolypeptide sequence comprising at least one amino acid residuedifference as compared to SEQ ID NO:26 or the functional fragmentthereof at one or more amino acid positions; and c) which exhibits animproved property selected from i) enhanced catalytic activity, ii)reduced sensitivity to proteolysis, iii) increased tolerance to acidicpH, iv) reduced aggregation, v) reduced immunogenicity, or a combinationof any of i), ii), iii), iv), or v), as compared to the referencesequence SEQ ID NO:26.

In some embodiments, the present invention also provides engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activitycomprising: a) an amino acid sequence having at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater sequence identity toreference sequence SEQ ID NO:26 or a functional fragment thereof; b) apolypeptide sequence comprising at least one amino acid residuedifference as compared to SEQ ID NO:26 or the functional fragmentthereof at one or more amino acid positions; and c) which exhibits animproved property selected from i) enhanced catalytic activity, ii)reduced sensitivity to proteolysis, iii) increased tolerance to acidicpH, iv) reduced aggregation, v) reduced immunogenicity, or a combinationof any of i), ii), iii), iv), or v), as compared to the referencesequence SEQ ID NO:26.

In some additional embodiments, the present invention providesengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity comprising: a) an amino acid sequence having at least 85%sequence identity to reference sequence SEQ ID NO:26 or a functionalfragment thereof; b) a polypeptide sequence comprising at least oneamino acid residue difference as compared to SEQ ID NO:26 or thefunctional fragment thereof at one or more amino acid positions; and c)which exhibits an improved property selected from i) enhanced catalyticactivity, ii) reduced sensitivity to proteolysis, iii) increasedtolerance to acidic pH, iv) reduced aggregation, v) reducedimmunogenicity, or a combination of any of i), ii), iii), iv), or v), ascompared to the reference sequence SEQ ID NO:26. In some embodiments,the engineered polypeptides having phenylalanine ammonia-lyase (PAL)activity of the present invention comprise an amino acid sequence havingat least 85% sequence identity to reference sequence SEQ ID NO:26, andat least one amino acid residue difference as compared to SEQ ID NO:126,and that exhibit at least one improved property selected from enhancedcatalytic activity, reduced sensitivity to proteolysis, increasedtolerance to acidic pH, reduced aggregation, and/or reducedimmunogenicity, as compared to SEQ ID NO:26. In some embodiments of theengineered polypeptides, the amino acid residue difference as comparedto SEQ ID NO:26 is selected from one or more of the followingsubstitutions or substitution sets A24E/G381L; L127V; A129I/V; S131C/T;H132L/S; R134C/F/H/K; R134H/Y378E/G381L; R134H/Y378E/G381L/V388T;R134H/V388T; A136K; A289S; M372L; H374G/M/Q; G381A/C/F/I/L/M/N/Q/S/T;A383C/M; V388C/T; L431M; and/or L563M.

In some embodiments, the engineered polypeptide having phenylalanineammonia lyase (PAL) activity of the present invention has at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity toreference sequence SEQ ID NO:4. In some embodiments, the engineeredpolypeptide having phenylalanine ammonia lyase (PAL) activity of thepresent invention has at least about 90% sequence identity to referencesequence SEQ ID NO:4, while in some further embodiments, the engineeredpolypeptide has at least about 95% sequence identity to referencesequence SEQ ID NO:4. In some embodiments, the engineered polypeptidehaving phenylalanine ammonia lyase (PAL) activity of the presentinvention has at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to reference sequence SEQ ID NO:4. In someembodiments, the engineered polypeptide having phenylalanine ammonialyase (PAL) activity of the present invention has at least 90% sequenceidentity to reference sequence SEQ ID NO:4, while in some furtherembodiments, the engineered polypeptide has at least 95% sequenceidentity to reference sequence SEQ ID NO:4. In some further embodiments,the engineered polypeptides comprise functional fragments ofpolypeptides (e.g., any of the variant provided in the Tables, herein)having phenylalanine ammonia lyase (PAL) activity of the presentinvention.

In some embodiments, the engineered polypeptide having phenylalanineammonia lyase (PAL) activity of the present invention has at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and/or 26. In someembodiments, the engineered polypeptide having phenylalanine ammonialyase (PAL) activity of the present invention has at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity toSEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and/or 26. In someembodiments, the engineered polypeptide having phenylalanineammonia-lyase (PAL) activity comprise an amino acid sequence having atleast about 90% sequence identity to SEQ ID NO:6, 8, 10, 12, 14, 16, 18,20, 22, 24, and/or 26. In some embodiments, the engineered polypeptidehaving phenylalanine ammonia-lyase (PAL) activity comprises an aminoacid sequence having at least about 99% sequence identity to SEQ IDNO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and/or 26, or a functionalfragment thereof. In some further embodiments, the engineeredpolypeptides comprise functional fragments of polypeptides (e.g.,functional fragments of SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24,and/or 26, as well as any of the variants provided in the Tables,herein) having phenylalanine ammonia lyase (PAL) activity of the presentinvention.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity, wherein the engineeredpolypeptides are variant PALs provided in any of Tables 2-1 through 2-5and/or Tables 9-1 through 9-7.

In some embodiments, the engineered polypeptide having phenylalanineammonia-lyase (PAL) activity is an Anabaena variabilis enzyme. In someadditional embodiments, the engineered polypeptides having phenylalanineammonia-lyase (PAL) activity are thermostable. In some embodiments, theengineered polypeptides having phenylalanine ammonia-lyase (PAL)activity are resistant to proteolysis. In some additional embodiments,the engineered polypeptides having phenylalanine ammonia-lyase (PAL)activity are resistant to proteolysis by at least one digestive tractenzyme. In some further embodiments, the engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity are resistant to proteolysisby chymotrypsin, trypsin, carboxypeptidases, and/or elastases. In somefurther embodiments, the engineered polypeptide having phenylalanineammonia-lyase (PAL) activity is acid stable.

The present invention also provides engineered polypeptides havingphenylalanine ammonia-lyase (PAL) activity that are deimmunized. In someembodiments, the deimmunized engineered polypeptides comprise an aminoacid sequence having at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or greater, sequence identity to SEQ ID NO:6, 8, 10, 12, 14,16, 18, 20, 22, 24, and/or 26. In some additional embodiments, thedeimmunized engineered polypeptides comprise an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or greater, sequence identity to SEQ ID NO:6, 8, 10, 12, 14, 16,18, 20, 22, 24, and/or 26. In some embodiments, the deimmunizedengineered polypeptides comprise an amino acid sequence having at least95% sequence identity to SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24,and/or 26. In some embodiments, the deimmunized engineered polypeptidescomprise an amino acid sequence having 95% sequence identity to SEQ IDNO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and/or 26.

In still some further embodiments, the present invention providespurified engineered polypeptides having phenylalanine ammonia lyase(PAL) activity.

The present invention also provides polynucleotide sequences encoding atleast one engineered polypeptide having phenylalanine ammonia lyase(PAL), as set forth herein. In some embodiments, the polynucleotidesequence is operably linked to a control sequence. In some additionalembodiments, the polynucleotide sequence is codon-optimized.

The present invention also provides expression vectors comprising atleast one polynucleotide sequence encoding at least one engineeredpolypeptide having phenylalanine ammonia-lyase (PAL) activity, asprovided herein. In some embodiments, the expression vector furthercomprises at least one control sequence. In some embodiments, thecontrol sequence is a promoter. In some additional embodiments, thepromoter is a heterologous promoter.

The present invention also provides host cells transformed with at leastone polynucleotide sequence encoding at least one engineeredpolypeptides having phenylalanine ammonia-lyase (PAL) activity, and/orat least one expression vector comprising at least one polynucleotidesequence encoding at least one engineered polypeptide havingphenylalanine ammonia-lyase (PAL) activity and at least one controlsequence. In some embodiments, the host cells comprise at least oneengineered polypeptide having phenylalanine ammonia-lyase (PAL) activitythat is codon-optimized. In some embodiments, the host cell is E. coli.

The present invention also provides methods of producing at least oneengineered PAL polypeptide in a host cell comprising culturing a hostcell comprising at least one polynucleotide encoding at least oneengineered polypeptide having phenylalanine ammonia-lyase (PAL)activity, and/or at least one expression vector comprising at least onepolynucleotide sequence encoding at least one engineered polypeptidehaving phenylalanine ammonia-lyase (PAL) activity and at least onecontrol sequence, under suitable culture conditions, such that theengineered PAL polypeptide is produced. In some embodiments, the methodsfurther comprise the step of recovering at least one engineeredpolypeptide having phenylalanine ammonia-lyase (PAL) from the cultureand/or host cells. In some further embodiments, the methods furthercomprise the step of purifying at least one engineered polypeptidehaving phenylalanine ammonia-lyase (PAL).

The present invention also provides compositions comprising at least oneengineered polypeptide having phenylalanine ammonia-lyase (PAL) activityas provided herein. In some embodiments, the composition is apharmaceutical composition. In some embodiments, the composition is adietary and/or nutritional supplement. In some further embodiments, thepharmaceutical compositions further comprise at least onepharmaceutically acceptable excipient and/or carrier. In some additionalembodiments, the composition is suitable for the treatment ofphenylketonuria. In some further embodiments, the pharmaceuticalcomposition is suitable for oral administration to a human. In someembodiments, the composition is in the form of a pill, tablet, capsule,gelcap, liquid, or emulsion. In yet some further embodiments, the pill,tablet, capsule, or gelcap further comprises an enteric coating. In someadditional embodiments, the pharmaceutical composition is suitable forparenteral injection into a human. In some embodiments, thepharmaceutical composition is coadministered with at least oneadditional therapeutically effective compound. In some furtherembodiments, the pharmaceutical composition comprises at least oneadditional therapeutically effective compound. In some additionalembodiments, the pharmaceutical composition is present in a dietaryand/or nutritional supplement.

The present invention also provides methods for treating and/orpreventing the symptoms of phenylketonuria in a subject, comprisingproviding a subject having phenylketonuria, and providing at least onecomposition provided herein to the subject. In some embodiments, thecomposition comprises a pharmaceutical composition, while in somealternative embodiments, the composition comprises a dietary/nutritionalsupplement. In some embodiments of the methods, the symptoms ofphenylketonuria are ameliorated. In some additional embodiments, thetreated subject is able to eat a diet that is less restricted in itsmethionine, phenylalanine, and/or tyrosine content than diets requiredby subjects exhibiting the symptoms of phenylalanine. In someembodiments, the treated subject (i.e., a subject who has been providedwith at least one composition comprising at least one engineeredpolypeptide having phenylalanine ammonia-lyase (PAL) activity asprovided herein) is able to eat a diet that is less restricted in itsmethionine, phenylalanine and/or tyrosine content than diets required bysubjects who have not been provided at least one composition as providedherein. In some embodiments, the composition provided to the subjectscomprises a pharmaceutical composition, while in some alternativeembodiments, the composition comprises a dietary/nutritional supplement.The present invention also provides treated subjects, wherein thesubject has been administered at least one composition and/orpharmaceutical composition comprising at least one engineeredpolypeptide having phenylalanine ammonia-lyase (PAL) activity asprovided herein. In some embodiments, the subject is an animal selectedfrom primates, rodents, and lagamorphs. In some additional embodiments,the subject is a mouse. In some further embodiments, the subject is ahuman. In still some further embodiments, the subject is a human infantor child, while in some alternative embodiments, the subject is a humanadult or young adult.

The present invention also provides uses of the compositions comprisingat least one engineered polypeptide having phenylalanine ammonia-lyase(PAL) activity provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of wild-type PAL protein sequences:Anabaena variabilis PAL (NCBI YP_(—) 324488.1 (SEQ ID NO:4)); Nostocpunctifonne phenylalanine/histidine ammonia lyase “NpPHAL” (NCBIYP_001865631.1 (SEQ ID NO:30)); Rivularia sp. histidine ammonia-lyase“RspHAL” (NCBI YP_007056096.1 (SEQ ID NO:31)); Oscillatoria sp.histidine ammonia-lyase “Osp HAL” (NCBI ZP_07108482.1 (SEQ ID NO:32));and Gloeocapsa sp. histidine ammonia-lyase “GspHAL” (NCBIYP_007127054.1) (SEQ ID NO:33)).

FIG. 2A shows the reduced sensitivity to proteolysis (expressed aschymotrypsin and trypsin tolerance) as compared to wild-type AvPALtested at pH 7.0 for Variant No. 22 (SEQ ID NO:8), Variant No. 30 (SEQID NO:6) and Variant No. 36 (SEQ ID NO:10) as further described inExample 4.

FIG. 2B provides a graph showing an increased tolerance to acidic pH ascompared to wild-type AvPAL tested at pH 4.0 to 5.2, for Variant Nos.22, 30 and 36 as further described in Example 4.

FIG. 3 provides K_(M) results for wild-type PAL and Variant No. 36.

FIG. 4 provides data showing the amino acid specificity of wild-type PALand Variant No. 36.

FIG. 5 provides results showing the relative stability of wild-type PALand Variant No. 36 exposed to human chymotrypsin and trypsin.

FIG. 6 provides results showing the relative stability of wild-type PAL,and Variant Nos. 36, 42, and 43 exposed to porcine pancreatic extract.

FIG. 7 provides results showing the effect of varying concentrations ofsodium taurocholate on the susceptibility of Variant 36 to proteolysis.

FIG. 8 provides results showing serum phenylalanine levels in micetreated with inactive protein (BSA), wild-type PAL, wild-type PAL incombination with aprotinin, or Variant 42.

DESCRIPTION OF THE INVENTION

The present invention provides engineered PAL polypeptides, mutants,biologically active fragments and analogues thereof, and pharmaceuticaland industrial compositions comprising the same.

The invention provides engineered phenylalanine ammonia-lyase (PAL)polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.In some embodiments, the engineered PAL polypeptides are optimized toprovide enhanced catalytic activity, as well as reduced sensitivity toproteolysis and increased tolerance to acidic pH levels. In someembodiments the engineered PAL polypeptides are deimmunized. Theinvention also relates to the use of the compositions comprising theengineered PAL polypeptides for therapeutic and industrial purposes.

Abbreviations and Definitions:

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the application as a whole. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by reference.

Also, as used herein, the singular “a”, “an,” and “the” include theplural references, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus,every numerical range disclosed herein is intended to encompass everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.It is also intended that every maximum (or minimum) numerical limitationdisclosed herein includes every lower (or higher) numerical limitation,as if such lower (or higher) numerical limitations were expresslywritten herein.

The term “about” means an acceptable error for a particular value. Insome instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of agiven value range. In some instances, “about” means within 1, 2, 3, or 4standard deviations of a given value.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the application as a whole. Accordingly, the terms definedimmediately below are more fully defined by reference to the applicationas a whole. Nonetheless, in order to facilitate understanding of theinvention, a number of terms are defined below.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

“EC” number refers to the Enzyme Nomenclature of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (NC-IUBMB). The IUBMB biochemical classification is a numericalclassification system for enzymes based on the chemical reactions theycatalyze.

“ATCC” refers to the American Type Culture Collection whosebiorepository collection includes genes and strains.

“NCBI” refers to National Center for Biological Information and thesequence databases provided therein.

As used herein, the term “phenylalanine ammonia-lyase (PAL) polypeptide”refers to a class of enzymes within the aromatic amino acid lyase family(EC 4.3.1.23, EC 4.3.1.24 and EC4.3.1.25) which also includes histidineammonia-lyase, and tyrosine ammonia-lyase. The PAL polypeptides are alsosometimes referred to as phenylalanine/tyrosine ammonia-lyases becausesome PAL enzymes may use tyrosine as well as phenylalanine as asubstrate. However, the AvPAL and variants disclosed and claimed hereindo not use tyrosine as a substrate. PAL polypeptides catalyze theconversion of L-phenylalanine to trans-cinnamic acid and ammonia. PALactivity refers to the enzymatic activity of PAL polypeptides. In somepreferred embodiments, a PAL enzyme also contains the cofactor3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). This cofactor mayberequired for catalytic activity and is formed by cyclization anddehydration of a conserved active site Ala167-Ser168-Gly169 tripeptidesegment.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation).

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.

The term “engineered,” “recombinant,” “non-naturally occurring,” and“variant,” when used with reference to a cell, a polynucleotide or apolypeptide refers to a material or a material corresponding to thenatural or native form of the material that has been modified in amanner that would not otherwise exist in nature or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

“Deimmunized” as used herein, refers to the manipulation of a protein tocreate a variant that is not as immunogenic as the wild-type orreference protein. In some embodiments, the deimmunization is complete,in that the variant protein does not stimulate an immune response inpatients to whom the variant protein is administered. This response canbe measured by various methods including but not limited to, thepresence or abundance of neutralizing (i.e., anti-drug antibodies), thepresence of an anaphylactic response, or the prevalence or intensity ofcytokine release upon administration of the protein. In someembodiments, the variant protein is less immunogenic than the wild-typeor reference protein. In some embodiments, deimmunization involvesmodifications to proteins (e.g., epitopes) that are recognized by T-cellreceptors. In some embodiments, the T-cell epitopes are removed from awild-type or reference protein in order to produce a deimmunized variantprotein. In some embodiments, the deimmunized protein shows lower levelsof response in biochemical and cell-biological predictors of humanimmunological responses including dendritic-cell T-cell activationassays, or human leukocyte antigen (HLA) peptide binding assays.

“Coding sequence” refers to that part of a nucleic acid (e.g., a gene)that encodes an amino acid sequence of a protein.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides and polypeptides, and are determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homologyalignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.Mol. Biol., 48:443 [1970]), by the search for similarity method ofPearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 [1988]), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin SoftwarePackage), or by visual inspection, as known in the art. Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity include, but are not limited to the BLAST andBLAST 2.0 algorithms (See e.g., Altschul et al., J. Mol. Biol., 215:403-410 [1990]; and Altschul et al., Nucleic Acids Res., 3389-3402[1977]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information website. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length “W” in the query sequence, whicheither match or satisfy some positive-valued threshold score “T,” whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (See, Altschul etal, supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters “M” (rewardscore for a pair of matching residues; always >0) and “N” (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity “X” from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915 [1989]). Exemplary determination of sequence alignment and %sequence identity can employ the BESTFIT or GAP programs in the GCGWisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, at least 100 residues in length or the fulllength of the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides or polypeptides over a “comparison window” to identifyand compare local regions of sequence similarity. In some embodiments, a“reference sequence” can be based on a primary amino acid sequence,where the reference sequence is a sequence that can have one or morechanges in the primary sequence. For instance, the phrase “referencesequence based on SEQ ID NO:4 having a valine at the residuecorresponding to X39” refers to a reference sequence in which thecorresponding residue at position X39 in SEQ ID NO:4 (e.g., an alanine),has been changed to valine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Corresponding to”, “reference to,” and “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refer to the numbering of the residues of a specified referencesequence when the given amino acid or polynucleotide sequence iscompared to the reference sequence. In other words, the residue numberor residue position of a given polymer is designated with respect to thereference sequence rather than by the actual numerical position of theresidue within the given amino acid or polynucleotide sequence. Forexample, a given amino acid sequence, such as that of an engineered PAL,can be aligned to a reference sequence by introducing gaps to optimizeresidue matches between the two sequences. In these cases, although thegaps are present, the numbering of the residue in the given amino acidor polynucleotide sequence is made with respect to the referencesequence to which it has been aligned.

“Amino acid difference” and “residue difference” refer to a differencein the amino acid residue at a position of a polypeptide sequencerelative to the amino acid residue at a corresponding position in areference sequence. The positions of amino acid differences generallyare referred to herein as “Xn,” where n refers to the correspondingposition in the reference sequence upon which the residue difference isbased. For example, a “residue difference at position X91 as compared toSEQ ID NO:4” refers to a difference of the amino acid residue at thepolypeptide position corresponding to position 91 of SEQ ID NO:4. Thus,if the reference polypeptide of SEQ ID NO:4 has a alanine at position91, then a “residue difference at position X91 as compared to SEQ IDNO:4” refers to an amino acid substitution of any residue other thanalanine at the position of the polypeptide corresponding to position 91of SEQ ID NO:4. In most instances herein, the specific amino acidresidue difference at a position is indicated as “XnY” where “Xn”specified the corresponding residue and position of the referencepolypeptide (as described above), and “Y” is the single letteridentifier of the amino acid found in the engineered polypeptide (i.e.,the different residue than in the reference polypeptide). In someinstances (e.g., in the Tables in the Examples), the present disclosurealso provides specific amino acid differences denoted by theconventional notation “AnB”, where A is the single letter identifier ofthe residue in the reference sequence, “n” is the number of the residueposition in the reference sequence, and B is the single letteridentifier of the residue substitution in the sequence of the engineeredpolypeptide. In some instances, a polypeptide of the present disclosurecan include one or more amino acid residue differences relative to areference sequence, which is indicated by a list of the specifiedpositions where residue differences are present relative to thereference sequence. In some embodiments, where more than one amino acidcan be used in a specific residue position of a polypeptide, the variousamino acid residues that can be used are separated by a “/” (e.g.,X307G/X307Q or X307G/Q). The present disclosure includes engineeredpolypeptide sequences comprising one or more amino acid differences thatinclude either/or both conservative and non-conservative amino acidsubstitutions.

The terms “amino acid substitution set” and “substitution set” refers toa group of amino acid substitutions within a polypeptide sequence. Insome embodiments, substitution sets comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or more amino acid substitutions. In someembodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant AvPAL polypeptideslisted in any of the Tables in the Examples. For example, thesubstitution set present in Variant 36 isA39V/A91V/N290G/H307G/L407V/T524S, wherein the amino acid positions arerelative to SEQ ID NO:4.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid (e.g., alanine, valine, leucine, and isoleucine); an amino acidwith hydroxyl side chain is substituted with another amino acid with ahydroxyl side chain (e.g., serine and threonine); an amino acids havingaromatic side chains is substituted with another amino acid having anaromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, andhistidine); an amino acid with a basic side chain is substituted withanother amino acid with a basis side chain (e.g., lysine and arginine);an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain (e.g., aspartic acid or glutamicacid); and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affect: (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine); (b) the charge or hydrophobicity; and/or (c) thebulk of the side chain. By way of example and not limitation, exemplarynon-conservative substitutions include an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered transaminase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. Insertions can be inthe internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

The terms “functional fragment” and “biologically active fragment” areused interchangeably herein, to refer to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full length engineered PAL of the present invention)and that retains substantially all of the activity of the full-lengthpolypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it (e.g.,protein, lipids, and polynucleotides). The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The recombinant PAL polypeptides may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, therecombinant PAL polypeptides provided herein are isolated polypeptides.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure PAL composition will compriseabout 60% or more, about 70% or more, about 80% or more, about 90% ormore, about 95% or more, and about 98% or more of all macromolecularspecies by mole or % weight present in the composition. In someembodiments, the object species is purified to essential homogeneity(i.e., contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species. In some embodiments, the isolated recombinantPAL polypeptides are substantially pure polypeptide compositions.

“Improved enzyme property” refers to an engineered PAL polypeptide thatexhibits an improvement in any enzyme property as compared to areference PAL polypeptide, such as a wild-type PAL polypeptide (e.g.,AvPAL wild-type having SEQ ID NO:4) or another engineered PALpolypeptide. Improved properties include but are not limited to suchproperties as increased protein expression, increased thermoactivity,increased thermostability, increased pH activity, increased stability,increased enzymatic activity, increased substrate specificity and/oraffinity, increased specific activity, increased resistance to substrateand/or end-product inhibition, increased chemical stability, improvedchemoselectivity, improved solvent stability, increased tolerance toacidic pH, increased tolerance to proteolytic activity (i.e., reducedsensitivity to proteolysis), reduced aggregation, increased solubility,reduced immunogenicity, and altered temperature profile.

“Increased enzymatic activity” and “enhanced catalytic activity” referto an improved property of the engineered PAL polypeptides, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) and/or an increase in percent conversionof the substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of PAL) as compared to the reference PAL enzyme (e.g.,wild-type AvPAL and/or another engineered AvPAL). Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.1 fold the enzymatic activity of the correspondingwild-type enzyme, to as much as 2-fold, 5-fold, 10-fold, 20-fold,25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or moreenzymatic activity than the naturally occurring PAL or anotherengineered PAL from which the PAL polypeptides were derived.

In some embodiments, the engineered PAL polypeptides have a k_(cat) ofat least 0.1/sec, at least 0.2/sec, at least 0.3/sec, at least 0.5/sec,at least 1.0/sec and in some preferred embodiments greater than 1.0/sec.In some embodiments, the K_(m) is in the range of about 1 μm to about 5mM; in the range of about 5 μm to about 2 mM; in the range of about 10nm to about 2 mM; or in the range of about 10 μm to about 1 mM. In somespecific embodiments, the engineered PAL enzyme exhibits improvedenzymatic activity in the range of 1.5 to 10 fold, 1.5 to 25 fold, 1.5to 50 fold, 1.5 to 100 fold or greater, than that of the reference PALenzyme. PAL activity can be measured by any standard assay known in theart, (e.g., by monitoring changes in spectrophotometric properties ofreactants or products). In some embodiments, the amount of productsproduced is measured by High-Performance Liquid Chromatography (HPLC)separation combined with UV absorbance or fluorescent detection directlyor following o-phthaldialdehyde (OPA) derivatization. In someembodiments, comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells, in order to minimizevariations in amount of enzyme produced by the host cells and present inthe lysates.

The term “improved tolerance to acidic pH” means that a recombinant PALaccording to the invention exhibits increased stability (i.e., higherretained activity at about pH 7.0, after exposure to acidic pH for aspecified period of time [1 hour, up to 24 hours]) as compared to areference PAL.

“Physiological pH” as used herein means the pH range generally found ina subject's (e.g., human) small intestine. There normally is a gradientpH from the pyloric valve to the large intestine, in the range of about6.0 to 7.5.

The term “acidic pH” used with reference to improved stability to acidicpH conditions or increased tolerance to acidic pH means a pH range ofabout 1.5 to 6.8.

The terms “proteolytic activity” and “proteolysis” used interchangeablyherein refer to the breakdown of proteins into smaller polypeptides oramino acids. The breakdown of proteins is generally the result ofhydrolysis of the peptide bond by protease (proteinase) enzymes.Protease enzymes include but are not limited to pepsin, trypsin,chymotrypsin, elastase; carboxypeptidase A and B, and peptidases (e.g.,amino peptidase, dipeptidase and enteropeptidase).

The phrases “reducing sensitivity to proteolysis” and “reducingproteolytic sensitivity” are used interchangeably herein mean that anengineered PAL polypeptide according to the invention will have a higherenzyme activity compared to a reference PAL in a standard assay (e.g.,as disclosed in the Examples) after treatment with one or moreproteases.

“Aggregation” means clumping or precipitation of a PAL polypeptide.Aggregation can lead to inactivation of the enzyme. The term “reducedaggregation” means an engineered PAL polypeptide will be less prone toaggregation, as compared to a reference PAL. Methods for assessingaggregation are known in the art, including but not limited to the useof fluorescent microscopy with appropriate dyes (e.g., thioflavin T orNile Red), dynamic light scattering, flow cytometry with appropriatedyes (e.g., Bodipy), filtration and analysis by SDS-PAGE, and/or Westernblotting, fluorescent correlation spectroscopy, and electron microscopy.There are commercially available kits to assess aggregation (e.g., theProteoStat® Protein Aggregation Assay kit [Enzo]).

“Conversion” refers to the enzymatic conversion (or biotransformation)of substrate(s) to the corresponding product(s). “Percent conversion”refers to the percent of the substrate that is converted to the productwithin a period of time under specified conditions. Thus, the “enzymaticactivity” or “activity” of a PAL polypeptide can be expressed as“percent conversion” of the substrate to the product in a specificperiod of time.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is more efficiently expressed in thatorganism. Although the genetic code is degenerate, in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thePAL enzymes are codon optimized for optimal production from the hostorganism selected for expression.

“Control sequence” refers herein to include all components that arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present disclosure. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, leaders,polyadenylation sequences, propeptide sequences, promoter sequences,signal peptide sequences, initiation sequences, and transcriptionterminators. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. In some embodiments, thecontrol sequences are provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide encoding a polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences that mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refers to those conditions in theenzymatic conversion reaction solution (e.g., ranges of enzyme loading,substrate loading, temperature, pH, buffers, co-solvents, etc.) underwhich a PAL polypeptide of the present disclosure is capable ofconverting a substrate to the desired product compound, Exemplary“suitable reaction conditions” are provided herein (See, the Examples).

“Loading”, such as in “compound loading” or “enzyme loading” refers tothe concentration or amount of a component in a reaction mixture at thestart of the reaction. “Substrate” in the context of an enzymaticconversion reaction process refers to the compound or molecule acted onby the PAL polypeptide. “Product” in the context of an enzymaticconversion process refers to the compound or molecule resulting from theaction of the PAL polypeptide on the substrate.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under suitable conditions using anysuitable medium (e.g., liquid, gel, or solid).

Recombinant polypeptides (e.g., PAL enzyme variants) can be producedusing any suitable methods known the art. For example, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Non-limiting examples of methods used for DNA and proteinengineering are provided in the following patents: U.S. Pat. Nos.6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393;7,783,428; and 8,383,346. After the variants are produced, they can bescreened for any desired property (e.g., high or increased activity, orlow or reduced activity, increased thermal activity, increased thermalstability, and/or acidic pH stability, etc.). In some embodiments,“recombinant PAL polypeptides” (also referred to herein as “engineeredPAL polypeptides,” “variant PAL enzymes,” and “PAL variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., a polynucleotide sequences encoding at least one AvPAL variant).In some embodiments, the host cells are prokaryotic or eukaryotic cellsthat have been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues include non-naturally occurring amino acid residues including,but not limited, to homoarginine, ornithine and norvaline, as well asnaturally occurring amino acids. In some embodiments, analogues alsoinclude one or more D-amino acid residues and non-peptide linkagesbetween two or more amino acid residues.

The term “therapeutic” refers to a compound administered to a subjectwho shows signs or symptoms of pathology having beneficial or desirablemedical effects.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject (e.g., human) comprising apharmaceutically effective amount of an engineered PAL polypeptideencompassed by the invention and an acceptable carrier.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

The term “subject” encompasses mammals such as humans, non-humanprimates, livestock, companion animals, and laboratory animals (e.g.,rodents and lagamorphs). It is intended that the term encompass femalesas well as males.

As used herein, the term “patient” means any subject that is beingassessed for, treated for, or is experiencing disease.

The term “infant” refers to a child in the period of the first monthafter birth to approximately one (1) year of age. As used herein, theterm “newborn” refers to child in the period from birth to the 28th dayof life. The term “premature infant” refers to an infant born after thetwentieth completed week of gestation, yet before full term, generallyweighing ˜500 to ˜2499 grams at birth. A “very low birth weight infant”is an infant weighing less than 1500 g at birth.

As used herein, the term “child” refers to a person who has not attainedthe legal age for consent to treatment or research procedures. In someembodiments, the term refers to a person between the time of birth andadolescence.

As used herein, the term “adult” refers to a person who has attainedlegal age for the relevant jurisdiction (e.g., 18 years of age in theUnited States). In some embodiments, the term refers to any fully grown,mature organism. In some embodiments, the term “young adult” refers to aperson less than 18 years of age, but who has reached sexual maturity.

As used herein, “composition” and “formulation” encompass productscomprising at least one engineered PAL of the present invention,intended for any suitable use (e.g., pharmaceutical compositions,dietary/nutritional supplements, feed, etc.).

The terms “administration” and “administering” a composition meanproviding a composition of the present invention to a subject (e.g., toa person suffering from the effects of PKU).

The term “carrier” when used in reference to a pharmaceuticalcomposition means any of the standard pharmaceutical carrier, buffers,and excipients, such as stabilizers, preservatives, and adjuvants.

The term “pharmaceutically acceptable” means a material that can beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents in which it is contained and that possesses the desiredbiological activity.

As used herein, the term “excipient” refers to any pharmaceuticallyacceptable additive, carrier, diluent, adjuvant, or other ingredient,other than the active pharmaceutical ingredient (API; e.g., theengineered PAL polypeptides of the present invention). Excipients aretypically included for formulation and/or administration purposes.

The term “therapeutically effective amount” when used in reference tosymptoms of disease/condition refers to the amount and/or concentrationof a compound (e.g., engineered PAL polypeptides) that ameliorates,attenuates, or eliminates one or more symptom of a disease/condition orprevents or delays the onset of symptom(s) (e.g., PKU). In someembodiments, the term is use in reference to the amount of a compositionthat elicits the biological (e.g., medical) response by a tissue,system, or animal subject that is sought by the researcher, physician,veterinarian, or other clinician.

The term “therapeutically effective amount” when used in reference to adisease/condition refers to the amount and/or concentration of acomposition that ameliorates, attenuates, or eliminates thedisease/condition.

It is intended that the terms “treating,” “treat” and “treatment”encompass preventative (e.g., prophylactic), as well as palliativetreatment.

Engineered PAL Polypeptides:

The parent PAL polypeptides from which the engineered PAL polypeptidesof the invention are derived from include bacterial strains such asAnabaena (e.g., A. variabilis), Nostoc (e.g., N. punctiforme),Rhodosporidium (e.g., R. toruloides), Streptomyces (e.g., S. maritimusor S. verticillatus), Oscillatoria sp., Gloeocapsa sp., and Rivulariasp. PAL enzymes from these strains have been identified and are wellknown. Homologous enzyme sequences from Anabaena (A. variabilis) ATCC29413 and NCBI YP_324488.1; Nostoc (N. punctiforme) ATCC 29133 and NCBIYP_00186563.1; Oscillatoria sp. PCC 6506 and NCBI ZP_(—) 07108482.1 andGloeocapsa sp. PCC7428 and NCBI YP_007127054.1 are provided in FIG. 1.The Nostoc punctiforme phenylalanine/histidine ammonia lyase “NpPHAL”(NCBI YP_001865631.1 (SEQ ID NO:30); Rivularia sp. histidineammonia-lyase “RspHAL” (NCBI YP_007056096.1 (SEQ ID NO:31); Oscillatoriasp. histidine ammonia-lyase “Osp HAL” (NCBI ZP_07108482.1 (SEQ IDNO:32); and Gloeocapsa sp. histidine ammonia-lyase “GspHAL” (NCBIYP_007127054.1) (SEQ ID NO:33) have more than 70% homology with AvPAL(SEQ ID NO:4).

Furthermore, when a particular PAL variant (i.e., an engineered PALpolypeptide) is referred to by reference to modification of particularamino acids residues in the sequence of a wild-type PAL or reference PALit is to be understood that variants of another PAL modified in theequivalent position(s) (as determined from the optional amino acidsequence alignment between the respective amino acid sequences) areencompassed herein. In some embodiments, the engineered PAL polypeptideis derived from any one of the polypeptides listed from the bacterialstrains above (i.e., Nostoc [N. punctifonne], Rhodosporidium [R.toruloides], Streptomyces [S. maritimus or S. verticillatus],Oscillatoria sp., Gloeocapsa sp and Rivularia sp.). In some additionalembodiments, the engineered PAL polypeptide of the present inventioncomprises the conserved active site Ala167-Ser168-Gly169 and comprisesat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto SEQ ID NO:4. In some embodiments, the engineered PAL polypeptidescomprise not only PAL activity but are also active on tyrosine and/orhistidine substrates.

In some embodiments, engineered PAL polypeptides are produced bycultivating a microorganism comprising at least one polynucleotidesequence encoding at least one engineered PAL polypeptide underconditions which are conducive for producing the engineered PALpolypeptide. In some embodiments, the engineered PAL polypeptide issubsequently recovered from the resulting culture medium and/or cells.

The present invention provides exemplary engineered PAL polypeptideshaving PAL activity. The Examples provide Tables showing sequencestructural information correlating specific amino acid sequence featureswith the functional activity of the engineered PAL polypeptides. Thisstructure-function correlation information is provided in the form ofspecific amino acid residue differences relative to the referenceengineered polypeptide of SEQ ID NO:4, as well as associatedexperimentally determined activity data for the exemplary engineered PALpolypeptides.

In some embodiments, the engineered PAL polypeptides of the presentinvention having PAL activity comprise a) an amino acid sequence havingat least 85% sequence identity to reference sequence SEQ ID NO:4; b) anamino acid residue difference as compared to SEQ ID NO:4 at one or moreamino acid positions; and c) which exhibits an improved propertyselected from i) enhanced catalytic activity, ii) reduced proteolyticsensitivity, iii) increased tolerance to acidic pH, iv) reducedaggregation or a combination of any of i), ii), iii) or iv) as comparedto the reference sequence.

In some embodiments the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:4, and an amino acid residuedifference as compared to SEQ ID NO:4, at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20or more amino acid positions compared to SEQ ID NO:4 or a sequencehaving at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO:4). In some embodiments, the residue difference ascompared to SEQ ID NO:4, at one or more positions includes at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acidsubstitutions. In some embodiments, the engineered PAL polypeptide is apolypeptide listed in the Tables provided in the Examples.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:4 and an amino acid residuedifference as compared to SEQ ID NO:4 at one or more amino acidpositions are selected from X39; X54; X59; X73; X91; X158; X112, X134,X180; X195; X240; X243; X245; X256; X257; X270; X290; X304, X305; X307;X308; X326; X349; X353; X364; X394; X399; X400; X404; X407; X443; X453;X459; X460; X463; X474; X509; X521; X522; X524; X528; X546; X564; or anycombination thereof, when optimally aligned with the amino acid sequenceof SEQ ID NO:4. In some embodiments the amino acid difference is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or greater amino acid positions.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85% (at least 88%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO:4 andcomprise an amino acid residue difference at position H307 andoptionally an amino acid residue difference at 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more amino acid positions. In some embodiments the amino acidresidue difference at position 307 is H307/G/Q/M.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85% (at least 88%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO:4 andcomprise at least one amino acid residue difference selected from acombination of one or more of A39; T54; G59, S73; A91; Y158; S180; K195;A112; R134; Q240; T243; I245; A256; L257; N270; N290; Y304; R305; H307;E308; I326; L349; D353; L364; A394; S399; N400; P404; L407; F443; N453;Y459; T460; T463; N474; E509; Q521; K522; T524; P528; S546; and/or P564.In some additional embodiments, there are amino acid residue differencesat 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions.

In some embodiments, the engineered PAL polypeptides exhibiting animproved property have at least 85% (at least 88%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO:4 andcomprise an amino acid residue difference selected from a combination ofone or more of A39V; T54K; G59R; S73K; A112C; R134Q; A91V; Y158H; S180A;K195E; Q240R/W; T243I/L; I245L; A256G; L257W/A; N270K; N290G; Y304H;R305M; H307G/Q/M; E308Q; I326F; L349M; D353A/N; L364Q; A394V; S399N;N400K; P404A; L407V; F443H; N453G; Y459F; T460G; T463N; N474Q; E509L;Q521K/S; K522Y/F/N; T524S; P528L; S546R; and P564 G/L/M, when optimallyaligned with SEQ ID NO:4.

In some embodiments, the amino acid residue difference is selected froma combination of one or more of A39V; A91V; A256G; N290G; A394V; S399N;P404A; L407V; K522Y/F/N; and/or T524S, when optimally aligned with SEQID NO:4.

In some embodiments, the present invention provides functional fragmentsof engineered PAL polypeptides. In some embodiments, functionalfragments comprise at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%of the activity of the engineered PAL polypeptide from which it wasderived (i.e., the parent engineered PAL). In some embodiments,functional fragments comprise at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% of the parent sequence of the engineered PAL. In someembodiments the functional fragment will be truncated by less than 5,less than 10, less than 15, less than 10, less than 25, less than 30,less than 35, less than 40, less than 45, and less than 50 amino acids.

In some embodiments, the present invention provides functional fragmentsof engineered PAL polypeptides. In some embodiments, functionalfragments comprise at least about 95%, 96%, 97%, 98%, or 99% of theactivity of the engineered PAL polypeptide from which it was derived(i.e., the parent engineered PAL). In some embodiments, functionalfragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% of the parent sequence of the engineered PAL. In some embodimentsthe functional fragment will be truncated by less than 5, less than 10,less than 15, less than 10, less than 25, less than 30, less than 35,less than 40, less than 45, less than 50, less than 55, less than 60,less than 65, or less than 70 amino acids.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property has at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater aminoacid sequence identity with SEQ ID NO:6 and an amino acid residuedifference as compared to SEQ ID NO:6, at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 ormore amino acid positions) compared to SEQ ID NO:6, or a sequence havingat least 85%, at least 88%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or greater amino acid sequence identity with SEQID NO:6. In some embodiments, the engineered PALs comprise at least 90%sequence identity to SEQ ID NO:6 and comprise an amino acid differenceas compared to SEQ ID NO:6 of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore amino acid positions. In some embodiments, the engineered PALpolypeptide consists of the sequence of SEQ ID NO:6.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater aminoacid sequence identity with SEQ ID NO:10, or a functional fragmentthereof and an amino acid residue difference as compared to SEQ IDNO:10, at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15 or more amino acid positions) compared toSEQ ID NO:10, or a sequence having at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or greateramino acid sequence identity with SEQ ID NO:10. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:10,and comprise an amino acid difference as compared to SEQ ID NO:10, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:10.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:12 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:12at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:12, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:12. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:12,and comprise an amino acid difference as compared to SEQ ID NO:12, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:12.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:14 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:14at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:14, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:14. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:14,and comprise an amino acid difference as compared to SEQ ID NO:14, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:14.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:16 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:16at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:16, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:16. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:16,and comprise an amino acid difference as compared to SEQ ID NO:16, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:16.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:18 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:18at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:18, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:18. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:18,and comprise an amino acid difference as compared to SEQ ID NO:18, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:18.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:20 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:20at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:20, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:20. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:20,and comprise an amino acid difference as compared to SEQ ID NO:20, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:20.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:22 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:22at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:22, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:22. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:22,and comprise an amino acid difference as compared to SEQ ID NO:22, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:22.

In some embodiments, the engineered PAL polypeptides exhibiting at leastone improved property have at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:24 or a functional fragmentthereof and an amino acid residue difference as compared to SEQ ID NO:24at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15 or more amino acid positions) compared to SEQ IDNO:24, or a sequence having at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO:24. In some embodiments, theengineered PALs comprise at least 95% sequence identity to SEQ ID NO:24,and comprise an amino acid difference as compared to SEQ ID NO:24, of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid positions. Insome embodiments, the engineered PAL polypeptide consists of thesequence of SEQ ID NO:24.

Variants with Reduced Sensitivity to Proteolysis:

In some embodiments, the engineered PAL polypeptides of the presentinvention have PAL activity, exhibit reduced sensitivity to proteolysis,and comprise: a) an amino acid sequence having at least 85% sequenceidentity to reference sequence SEQ ID NO:4; an b) an amino acid residuedifference as compared to SEQ ID NO:4 at one or more amino acidpositions.

In some embodiments, the engineered PAL polypeptides that exhibitreduced sensitivity to proteolysis have at least 85%, at least 88%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater amino acid sequence identity with SEQ ID NO:4 and an amino acidresidue difference as compared to SEQ ID NO:4 at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20or more amino acid positions compared to SEQ ID NO:4 or a sequencehaving at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO:4).

In some embodiments, the engineered PAL polypeptides that exhibitreduced sensitivity to proteolysis have at least 85%, at least 88%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater amino acid sequence identity with SEQ ID NO:4 and an amino acidresidue difference as compared to SEQ ID NO:4, at one or more amino acidpositions are selected from X39; X54; X59; X73; X91; X158; X112, X134,X180; X195; X240; X243; X245; X256; X257; X270; X290; X304, X305; X307;X308; X326; X349; X353; X364; X394; X399; X400; X404; X407; X443; X453;X459; X460; X463; X474; X509; X521; X522; X524; X528; X546; X564; or anycombination thereof, when optimally aligned with the amino acid sequenceof SEQ ID NO: 4. In some embodiments the amino acid difference is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or greater amino acid positions.

In some embodiments, the engineered PAL polypeptides that exhibitreduced sensitivity to proteolysis have at least 85%, at least 88%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQID NO:4, and comprise an amino acid residue difference at position X307;X326; X460; X307; and/or X528 and optionally an amino acid residuedifference at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidpositions. In some embodiments, the amino acid residue difference isselected from Y304H/W; R305L/M; H307G/M/Q; I326F; Q240W; T460G; P528L;and any of these substitutions in combination, when aligned with SEQ IDNO:4.

In some embodiments, the engineered PAL polypeptides that exhibitreduced sensitivity to proteolysis have at least 85%, at least 88%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater amino acid sequence identity with any of SEQ ID NOS:10, 12, 14,16, 18, 20, 22, and/or 24, or a functional fragment thereof and an aminoacid residue difference as compared to SEQ ID NOS:10, 12, 14, 16, 18,20, 22, and/or 24, at one or more amino acid positions (such as at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more amino acid positions)compared to SEQ ID NOS:10, 12, 14, 16, 18, 20, 22, and/or 24, or asequence having at least 85%, at least 88%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or greater amino acid sequenceidentity with SEQ ID NOS:10, 12, 14, 16, 18, 20, 22, and/or 24. In someembodiments, the engineered PAL comprises at least 95% sequence identityto SEQ ID NOS:10, 12, 14, 16, 18, 20, 22, and/or 24, and comprise anamino acid difference as compared to SEQ ID NOS:10, 12, 14, 16, 18, 20,22, and/or 24, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacid positions. In some embodiments, the PAL comprises or consists ofthe sequence of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, and/or 24.

In some embodiments, the proteolytic sensitivity of the engineered PALpolypeptides is reduced by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, or atleast 95%, of that of the wild-type PAL (e.g., AvPAL having SEQ ID NO:4)or as compared to a reference PAL polypeptide under essentially the sameconditions. The proteolytic activity can be measured using any suitablemethods known in the art, including but not limited to those describedin the Examples.

In some embodiments, the engineered PAL polypeptides having reducedsensitivity to proteolysis have reduced sensitivity to a compositioncomprising one or more proteases, including, but not limited to pepsin,trypsin, chymotrypsin, carboxypeptidase A and B, peptidases (e.g., aminopeptidase, dipeptidase and enteropeptidase) when both the reference PALand the engineered PAL having the reduced sensitivity are compared andexposed to essentially the same amount and kind of protease underessentially the same conditions.

In some embodiments, the engineered PAL polypeptide having reducedsensitivity to proteolysis have enzyme activity levels that are about1.0 fold, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold,100-fold, 150-fold, 200-fold or more of the enzymatic activity of thereference PAL (e.g., AvPAL). In some embodiments, the engineeredpolypeptides have more enzyme activity, as compared to a reference PAL,when activity is measured at a pH range of 4.5 to 7.5; when activity ismeasured at a pH range of 4.5 to 6.5; when activity is measured at a pHrange of 5.0 to 7.5; when activity is measured at a pH range of 5.0 to6.5; when activity is measured at a pH range of 5.5 to 7.5; and/or alsowhen activity is measured at a pH range of 5.5 to 6.5. In some otherembodiments, the engineered PAL polypeptides have K_(m) values in therange of 1 μM to 5 mM.

Variants with Increased Tolerance to Acidic pH:

In some embodiments, the engineered PAL polypeptides of the inventionhave PAL activity, are tolerant to acidic pH levels and comprise: a) anamino acid sequence having at least 85% sequence identity to referencesequence SEQ ID NO:4, or a fragment thereof; and b) an amino acidresidue difference as compared to SEQ ID NO:4, at one or more amino acidpositions.

In some embodiments, the engineered PAL polypeptides that exhibitincreased tolerance to acidic pH as compared to wild-type AvPAL and/oranother reference polypeptide have at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or greateramino acid sequence identity with SEQ ID NO:4 and an amino acid residuedifference as compared to SEQ ID NO:4, at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20or more amino acid positions compared to SEQ ID NO:4, or a sequencehaving at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or greater amino acid sequence identitywith SEQ ID NO:4.

In some embodiments, the engineered PAL polypeptides that exhibitincreased tolerance to acidic pH as compared to wild-type AvPAL and/oranother reference polypeptide, have at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or greateramino acid sequence identity with SEQ ID NO:4, and an amino acid residuedifference as compared to SEQ ID NO:4, at one or more amino acidpositions are selected from X39; X54; X59; X73; X91; X158; X112, X134,X180; X195; X240; X243; X245; X256; X257; X270; X290; X304, X305; X307;X308; X326; X349; X353; X364; X394; X399; X400; X404; X407; X443; X453;X459; X460; X463; X474; X509; X521; X522; X524; X528; X546; X564; or anycombination thereof when optimally aligned with the amino acid sequenceof SEQ ID NO:4. In some embodiments the amino acid difference is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or greater amino acid positions.

In some embodiments, the engineered PAL polypeptides that exhibitincreased tolerance to acidic pH as compared to wild-type AvPAL and/oranother reference polypeptide have at least 85%, at least 88%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO:4, and comprise an amino acid residue difference at position X39;X54; X59; X73; X91; X158; X112, X134, X180; X195; X240; X243; X245;X256; X257; X270; X290; X304, X305; X307; X308; X326; X349; X353; X364;X394; X399; X400; X404; X407; X443; X453; X459; X460; X463; X474; X509;X521; X522; X524; X528; X546; X564; or any combination thereof; andoptionally an amino acid residue difference at 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more amino acid positions. In some embodiments the amino acidresidue difference is A39; T54; G59, S73; A91; Y158; S180; K195; A112;R134; Q240; T243; I245; A256; L257; N270; N290; Y304; R305; H307; E308;I326; L349; D353; L364; A394; S399; N400; P404; L407; F443; N453; Y459;T460; T463; N474; E509; Q521; K522; T524; P528; S546; and/or P564, whenaligned with SEQ ID NO:4. In some embodiments, the engineered PALpolypeptides that exhibit increased tolerance to acidic pH have at least85%, at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:4, and comprise an amino acid residuedifference at one or more positions A39V; T54K; G59R; S73K; A112C;R134Q; A91V; Y158H; S180A; K195E; Q240R/W; T243I/L; I245L; A256G;L257W/A; N270K; N290G; Y304H; R305M; H307G/Q/M; E308Q; I326F; L349M;D353A/N; L364Q; A394V; S399N; N400K; P404A; L407V; F443H; N453G; Y459F;T460G; T463N; N474Q; E509L; Q521K/S; K522Y/F/N; T524S; P528L; S546R;and/or P564 G/L/M; when aligned with SEQ ID NO:4.

In some embodiments, when all other assay conditions are essentially thesame, the engineered PAL polypeptides having increased tolerance toacidic pH as compared to a reference PAL polypeptide have an increasedtolerance at a pH range between 1.5 to 6.5; between 1.5 and 5.0; between2.0 to 5.5; between 3.0 and 6.8; between 3.0 and 5.5; between 4.0 and6.5; between 4.0 and 4.5; between 4.5 and between 5.0; between 4.5 and5.5, between 4.5 and 6.0; between 4.5 and 6.5; between 5.0 and 6.5;between 5.0 and 6.0; between 5.0 and 5.5; between 5.5 and 6.0; between6.0 and 6.5; and/or between 6.5 and 7.0. In some embodiments, theincreased tolerance to acidic pH is exhibited at a pH of about 3.5, 4.0,4.5, 5.0, 5.5, 6.0 and/or 6.5.

In some embodiments, the engineered PAL polypeptides that have increasedtolerance to acidic pH also exhibit greater PAL activity as compared toa reference PAL when measure by a standard assay. Any suitable assayfinds use in the present invention, including, but not limited to thoseprovided herein.

It is further contemplated that any of the exemplary engineeredpolypeptides (i.e., Variant No. 1-Variant No. 1010) find use as thestarting amino acid sequence for synthesizing other engineered PALpolypeptides, for example by subsequent rounds of evolution by addingnew combinations of various amino acid differences from otherpolypeptides and other residue positions described herein. In someembodiments, additional improvements are generated by including aminoacid differences at residue positions that were maintained as unchangedthroughout earlier rounds of evolution. It is not intended that thepresent invention be limited to any particular method for producingengineered PAL polypeptides, as any suitable method finds use, includingbut not limited to the methods provided herein.

Polynucleotides Encoding Engineered Polypeptides, Expression Vectors andHost Cells:

The present invention provides polynucleotides encoding the engineeredPAL polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing at least oneheterologous polynucleotide encoding the engineered PAL polypeptide(s)is introduced into appropriate host cells to express the correspondingPAL polypeptide(s).

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode an engineered PAL polypeptide. Thus, the presentinvention provides methods and compositions for the production of eachand every possible variation of PAL polynucleotides that could be madethat encode the PAL polypeptides described herein by selectingcombinations based on the possible codon choices, and all suchvariations are to be considered specifically disclosed for anypolypeptide described herein, including the amino acid sequencespresented in the Examples (e.g., in the various Tables).

In some embodiments, the codons are preferably optimized for utilizationby the chosen host cell for protein production. For example, preferredcodons used in bacteria are typically used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredPAL polypeptides contain preferred codons at about 40%, 50%, 60%, 70%,80%, 90%, or greater than 90% of the codon positions in the full lengthcoding region.

In some embodiments, the PAL polynucleotide encodes an engineeredpolypeptide having PAL activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to a reference sequence selected from SEQ IDNOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and/or 23, or the amino acidsequence of any variant (e.g., those provided in the Examples), and oneor more residue differences as compared to the reference polynucleotideof SEQ ID NOs:3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and/or 23, or the aminoacid sequence of any variant as disclosed in the Examples (for example1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). Insome embodiments, the reference sequence is selected from SEQ ID NOS:3,5, 7, 9, 11, 13, 15, 17, 19, 21, and/or 23.

In some embodiments, the PAL polynucleotide encodes an engineeredpolypeptide having PAL activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to reference sequence SEQ ID NO:4and one or more residue differences as compared to SEQ ID NO:4 atresidue positions from X39; X54; X59; X73; X91; X158; X112, X134, X180;X195; X240; X243; X245; X256; X257; X270; X290; X304, X305; X307; X308;X326; X349; X353; X364; X394; X399; X400; X404; X407; X443; X453; X459;X460; X463; X474; X509; X521; X522; X524; X528; X546; and/or X564; whenoptimally aligned with the polypeptide of SEQ ID NO:4.

In some embodiments, the polynucleotide encoding the engineered PALpolypeptides comprises a polynucleotide sequence selected from apolynucleotide sequence selected from SEQ ID NOS:3, 5, 7, 9, 11, 13, 15,17, 19, 21, and/or 23. In some embodiments, the polynucleotide encodingan engineered PAL polypeptide has at least 80%, 85%, 90%, 93%, 95%, 96%,97%, 98%, 99% nucleotide residue identity to SEQ ID NOS:2, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, and/or 23.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NOS:2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and/or23, or a complement thereof, or a polynucleotide sequence encoding anyof the variant PAL polypeptides provided herein. In some embodiments,the polynucleotide capable of hybridizing under highly stringentconditions encodes a PAL polypeptide comprising an amino acid sequencethat has one or more residue differences as compared to SEQ ID NO:4, atresidue positions selected from X39; X54; X59; X73; X91; X158; X112,X134, X180; X195; X240; X243; X245; X256; X257; X270; X290; X304, X305;X307; X308; X326; X349; X353; X364; X394; X399; X400; X404; X407; X443;X453; X459; X460; X463; X474; X509; X521; X522; X524; X528; X546; and/orX564.

In some embodiments, an isolated polynucleotide encoding any of theengineered PAL polypeptides herein is manipulated in a variety of waysto facilitate expression of the PAL polypeptide. In some embodiments,the polynucleotides encoding the PAL polypeptides comprise expressionvectors where one or more control sequences is present to regulate theexpression of the PAL polynucleotides and/or polypeptides. Manipulationof the isolated polynucleotide prior to its insertion into a vector maybe desirable or necessary depending on the expression vector utilized.Techniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art. In someembodiments, the control sequences include among others, promoters,leader sequences, polyadenylation sequences, propeptide sequences,signal peptide sequences, and transcription terminators. In someembodiments, suitable promoters are selected based on the host cellsselection. For bacterial host cells, suitable promoters for directingtranscription of the nucleic acid constructs of the present disclosure,include, but are not limited to promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff etal., Proc. Natl Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tacpromoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25[1983]). Exemplary promoters for filamentous fungal host cells, include,but are not limited to promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO96/00787), as well as the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitabletranscription terminator sequence (i.e., a sequence recognized by a hostcell to terminate transcription). In some embodiments, the terminatorsequence is operably linked to the 3′ terminus of the nucleic acidsequence encoding the PAL polypeptide. Any suitable terminator which isfunctional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are known in the art (See e.g., Romanoset al., supra).

In some embodiments, the control sequence is also a suitable leadersequence (i.e., a non-translated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequenceis operably linked to the 5′ terminus of the nucleic acid sequenceencoding the PAL polypeptide. Any suitable leader sequence that isfunctional in the host cell of choice find use in the present invention.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylationsequence (i.e., a sequence operably linked to the 3′ terminus of thenucleic acid sequence and which, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribedmRNA). Any suitable polyadenylation sequence which is functional in thehost cell of choice finds use in the present invention. Exemplarypolyadenylation sequences for filamentous fungal host cells include, butare not limited to the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase. Useful polyadenylation sequences for yeast hostcells are known (See e.g., Guo and Sherman, Mol. Cell. Bio.,15:5983-5990 [1995]).

In some embodiments, the control sequence is also a signal peptide(i.e., a coding region that codes for an amino acid sequence linked tothe amino terminus of a polypeptide and directs the encoded polypeptideinto the cell's secretory pathway). In some embodiments, the 5′ end ofthe coding sequence of the nucleic acid sequence inherently contains asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, in some embodiments, the 5′ end of thecoding sequence contains a signal peptide coding region that is foreignto the coding sequence. Any suitable signal peptide coding region whichdirects the expressed polypeptide into the secretory pathway of a hostcell of choice finds use for expression of the engineeredpolypeptide(s). Effective signal peptide coding regions for bacterialhost cells are the signal peptide coding regions include, but are notlimited to those obtained from the genes for Bacillus NCIB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Insome embodiments, effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells include, but are not limited to those from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

In some embodiments, the control sequence is also a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from any suitable source, including, butnot limited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those that cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention is directed to a recombinantexpression vector comprising a polynucleotide encoding an engineered PALpolypeptide, and one or more expression regulating regions such as apromoter and a terminator, a replication origin, etc., depending on thetype of hosts into which they are to be introduced. In some embodiments,the various nucleic acid and control sequences described herein arejoined together to produce recombinant expression vectors which includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the PAL polypeptideat such sites. Alternatively, in some embodiments, the nucleic acidsequence of the present invention is expressed by inserting the nucleicacid sequence or a nucleic acid construct comprising the sequence intoan appropriate vector for expression. In some embodiments involving thecreation of the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and bring about the expression of the PAL polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector is one in which, when introduced into the host cell, it isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, in someembodiments, a single vector or plasmid, or two or more vectors orplasmids which together contain the total DNA to be introduced into thegenome of the host cell, and/or a transposon is utilized.

In some embodiments, the expression vector contains one or moreselectable markers, which permit easy selection of transformed cells. A“selectable marker” is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in filamentous fungal host cells include, but are notlimited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae),argB (ornithine carbamoyltransferases), bar (phosphinothricinacetyltransferase; e.g., from S. hygroscopicus), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A.orzyae), sC (sulfate adenyltransferase), and trpC (anthranilatesynthase), as well as equivalents thereof. In another aspect, thepresent invention provides a host cell comprising at least onepolynucleotide encoding at least one engineered PAL polypeptide of thepresent invention, the polynucleotide(s) being operatively linked to oneor more control sequences for expression of the engineered PAL enzyme(s)in the host cell. Host cells suitable for use in expressing thepolypeptides encoded by the expression vectors of the present inventionare well known in the art and include but are not limited to, bacterialcells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Exemplary host cells also include various Escherichia colistrains (e.g., W3110 (ΔfhuA) and BL21).

Accordingly, in another aspect, the present invention provides methodsof producing the engineered PAL polypeptides, where the methods compriseculturing a host cell capable of expressing a polynucleotide encodingthe engineered PAL polypeptide under conditions suitable for expressionof the polypeptide. In some embodiments, the methods further comprisethe steps of isolating and/or purifying the PAL polypeptides, asdescribed herein.

Appropriate culture media and growth conditions for host cells are wellknown in the art. It is contemplated that any suitable method forintroducing polynucleotides for expression of the PAL polypeptides intocells will find use in the present invention. Suitable techniquesinclude, but are not limited to electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

Engineered PAL polypeptides with the properties disclosed herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring or engineered PAL polypeptide to any suitable mutagenesisand/or directed evolution methods known in the art, and/or as describedherein. An exemplary directed evolution technique is mutagenesis and/orDNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746). Otherdirected evolution procedures that can be used include, among others,staggered extension process (StEP), in vitro recombination (See e.g.,Zhao et al., Nat. Biotechnol., 16:258-261 [1998]), mutagenic PCR (Seee.g., Caldwell et al., PCR Methods Appl., 3:S136-S140 [1994]), andcassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA93:3525-3529 [1996]).

Mutagenesis and directed evolution methods can be readily applied toPAL-encoding polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Any suitable mutagenesis and directedevolution methods find use in the present invention and are well knownin the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,830,721, 6,132,970,6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861,6,297,053, 6,576,467, 6,444,468, 5,811238, 6,117,679, 6,165,793,6,180,406, 6,291,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065,6,506,603, 6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497,7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702,6,391,552, 6,358,742, 6,482,647, 6,335,160, 6,653,072, 6,355,484,6,03,344, 6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182,6,406,855, 6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391,640,6,309,883, 7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631,6,528,311, 6,287,862, 6,335,198, 6,352,859, 6,379,964, 7,148,054,7,629,170, 7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745,6,436,675, 6,961,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428,7,747,391, 7,747,393, 7,751,986, 6,376,246, 6,426,224, 6,423,542,6,479,652, 6,319,714, 6,521,453, 6,368,861, 7,421,347, 7,058,515,7,024,312, 7,620,502, 7,853,410, 7,957,912, 7,904,249, and all relatednon-US counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997];Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev.Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201[1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell,38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull etal., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat.Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291[1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang etal., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al.,Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391[1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO01/75767; WO 2009/152336, and U.S. Pat. No. 6,537,746, all of which areincorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzyme preparations to adefined temperature (or other assay conditions) and measuring the amountof enzyme activity remaining after heat treatments or other suitableassay conditions. Clones containing a polynucleotide encoding a PALpolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

For engineered polypeptides of known sequence, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods. In some embodiments, fragments ofup to about 100 bases can be individually synthesized, then joined(e.g., by enzymatic or chemical litigation methods, or polymerasemediated methods) to form any desired continuous sequence. For example,polynucleotides and oligonucleotides disclosed herein can be prepared bychemical synthesis using the classical phosphoramidite method (See e.g.,Beaucage et al., Tet. Lett., 22:1859-69 [1981]; and Matthes et al., EMBOJ., 3:801-05 [1984]), as it is typically practiced in automatedsynthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors).

Accordingly, in some embodiments, a method for preparing the engineeredPAL polypeptide can comprise: (a) synthesizing a polynucleotide encodinga polypeptide comprising an amino acid sequence selected from the aminoacid sequence of any variant as described herein, and (b) expressing thePAL polypeptide encoded by the polynucleotide. In some embodiments ofthe method, the amino acid sequence encoded by the polynucleotide canoptionally have one or several (e.g., up to 3, 4, 5, or up to 10) aminoacid residue deletions, insertions and/or substitutions. In someembodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25,1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residuedeletions, insertions and/or substitutions. In some embodiments, thesubstitutions are conservative or non-conservative substitutions.

The expressed engineered PAL polypeptide can be evaluated for anydesired improved property or combination of properties (e.g., activity,selectivity, stability, acid tolerance, protease sensitivity, etc.)using any suitable assay known in the art, including but not limited tothe assays and conditions described herein.

In some embodiments, any of the engineered PAL polypeptides expressed ina host cell are recovered from the cells and/or the culture medium usingany one or more of the well-known techniques for protein purification,including, among others, lysozyme treatment, sonication, filtration,salting-out, ultra-centrifugation, and chromatography.

Chromatographic techniques for isolation of the PAL polypeptidesinclude, among others, reverse phase chromatography, high-performanceliquid chromatography, ion-exchange chromatography,hydrophobic-interaction chromatography, size-exclusion chromatography,gel electrophoresis, and affinity chromatography. Conditions forpurifying a particular enzyme depends, in part, on factors such as netcharge, hydrophobicity, hydrophilicity, molecular weight, molecularshape, etc., and will be apparent to those having skill in the art. Insome embodiments, affinity techniques may be used to isolate theimproved PAL enzymes. For affinity chromatography purification, anyantibody that specifically binds a PAL polypeptide of interest may finduse. For the production of antibodies, various host animals, includingbut not limited to rabbits, mice, rats, etc., are immunized by injectionwith a PAL polypeptide, or a fragment thereof. In some embodiments, thePAL polypeptide or fragment is attached to a suitable carrier, such asBSA, by means of a side chain functional group or linkers attached to aside chain functional group.

In some embodiments, the engineered PAL polypeptide is produced in ahost cell by a method comprising culturing a host cell (e.g., an E. colistrain) comprising a polynucleotide sequence encoding an engineered PALpolypeptide as described herein under conditions conducive to theproduction of the engineered PAL polypeptide and recovering theengineered PAL polypeptide from the cells and/or culture medium. In someembodiments, the host cell produces more than one engineered PALpolypeptide.

In some embodiments, the present invention provides a method ofproducing an engineered PAL polypeptide comprising culturing arecombinant bacterial cell comprising a polynucleotide sequence encodingan engineered PAL polypeptide having at least 85%, 90%, 95%, 96%, 97%,98%, or 99% sequence identity to reference sequences SEQ ID NO:4, andone or more amino acid residue differences as compared to SEQ ID NO:4selected from X39; X91; X158; X180; X195; X243; X245; X256; X257; X270;X290; X307; X308; X326; X349; X364; X394; X399; X400; X404; X407; X443;X453; X459; X460; X463; X474; X522; X524; and X528, or combinationsthereof, when optimally aligned with the amino acid sequence of SEQ IDNO:4 under suitable culture conditions to allow the production of theengineered PAL polypeptide and optionally recovering the engineered PALpolypeptide from the culture and/or cultured bacterial cells. In someembodiments, the host cell produces more than one engineered PALpolypeptide.

In some embodiments, once the engineered PAL polypeptides are recoveredfrom the recombinant host cells and/or culture medium, they are furtherpurified by any suitable method(s) known in the art. In some additionalembodiments, the purified TAL polypeptides are combined with otheringredients and compounds to provide compositions and formulationscomprising the engineered PAL polypeptide as appropriate for differentapplications and uses (e.g., pharmaceutical compositions).

Compositions:

The present invention provides engineered PAL polypeptides suitable foruse in numerous compositions. These compositions find use in manyfields, including but not limited to pharmaceuticals,dietary/nutritional supplements, food, feed, and fine chemicalproduction. For example, in some embodiments, the present inventionprovides food and/or feeds comprising at least one engineered PALvariant and/or at least one polynucleotide sequence encoding at leastone PAL variant. In some embodiments, the present invention providesbeverages comprising at least one engineered PAL variant.

In some embodiments, the engineered PAL variant in food, feed, and/ornutritional/dietary supplement is glycosylated. Furthermore, theengineered PAL variants find use in any suitable edible enzyme deliverymatrix. In some embodiments, the engineered PAL variants are present inan edible enzyme delivery matrix designed for rapid dispersal of the PALvariant within the digestive tract of an animal upon ingestion of thevariant.

The present invention also provides engineered PAL polypeptides suitablefor use in production of fine chemicals and other industrially importantcompounds (See e.g., US Pat. Appln. Nos. 2013/0340119, 2013/0005012, and2005/0260724, and WO 2012/122333).

Pharmaceutical and Other Compositions

The present invention provides engineered PAL polypeptides suitable foruse in pharmaceutical and other compositions, such asdietary/nutritional supplements.

Depending on the mode of administration, these compositions comprising atherapeutically effective amount of an engineered PAL according to theinvention are in the form of a solid, semi-solid, or liquid. In someembodiments, the compositions include other pharmaceutically acceptablecomponents such as diluents, buffers, excipients, salts, emulsifiers,preservatives, stabilizers, fillers, and other ingredients. Details ontechniques for formulation and administration are well known in the artand described in the literature.

In some embodiments, the engineered PAL polypeptides are formulated foruse in oral pharmaceutical compositions. Any suitable format for use indelivering the engineered PAL polypeptides find use in the presentinvention, including but not limited to pills, tablets, gel tabs,capsules, lozenges, dragees, powders, soft gels, sol-gels, gels,emulsions, implants, patches, sprays, ointments, liniments, creams,pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks,suspensions (including but not limited to oil-based suspensions, oil-inwater emulsions, etc.), slurries, syrups, controlled releaseformulations, suppositories, etc. In some embodiments, the engineeredPAL polypeptides are provided in a format suitable for injection (i.e.,in an injectable formulation). In some embodiments, the engineered PALpolypeptides are provided in biocompatible matrices such as sol-gels,including silica-based (e.g., oxysilane) sol-gels. In some embodiments,the engineered PAL polypeptides are encapsulated. In some alternativeembodiments, the engineered PAL polypeptides are encapsulated innanostructures (e.g., nanotubes, nanotubules, nanocapsules, ormicrocapsules, microspheres, liposomes, etc.). Indeed, it is notintended that the present invention be limited to any particulardelivery formulation and/or means of delivery. It is intended that theengineered PAL polypeptides be administered by any suitable means knownin the art, including but not limited to parenteral, oral, topical,transdermal, intranasal, intraocular, intrathecal, via implants, etc.

In some embodiments, the engineered PAL polypeptides are chemicallymodified by glycosylation, pegylation (i.e., modified with polyethyleneglycol [PEG] or activated PEG, etc.) or other compounds (See e.g.,Ikeda, Amino Acids 29:283-287 [2005]; U.S. Pat. Nos. 7,531,341,7,534,595, 7,560,263, and 7,53,653; US Pat. Appln. Publ. Nos.2013/0039898, 2012/0177722, etc.). Indeed, it is not intended that thepresent invention be limited to any particular delivery method and/ormechanism.

In some additional embodiments, the engineered PAL polypeptides areprovided in formulations comprising matrix-stabilized enzyme crystals.In some embodiments, the formulation comprises a cross-linkedcrystalline engineered PAL enzyme and a polymer with a reactive moietythat adheres to the enzyme crystals. The present invention also providesengineered PAL polypeptides in polymers.

In some embodiments, compositions comprising the engineered PALpolypeptides of the present invention include one or more commonly usedcarrier compounds, including but not limited to sugars (e.g., lactose,sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice,potato, or other plant starch), cellulose (e.g., methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums(e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin,collagen, etc.). Additional components in oral formulations may includecoloring and or sweetening agents (e.g., glucose, sucrose, and mannitol)and lubricating agents (e.g., magnesium stearate), as well as entericcoatings (e.g., methacrylate polymers, hydroxyl propyl methyl cellulosephthalate, and/or any other suitable enteric coating known in the art).In some embodiments, disintegrating or solubilizing agents are included(e.g., cross-linked polyvinyl pyrrolidone, agar, alginic acid or saltsthereof, such as sodium alginate). In some embodiments, the engineeredPAL polypeptide are combined with various additional components,including but not limited to preservatives, suspending agents,thickening agents, wetting agents, alcohols, fatty acids, and/oremulsifiers, particularly in liquid formulations.

In some embodiments, the engineered PAL polypeptide are be combined withvarious additional components, including but not limited topreservatives, suspending agents, thickening agents, wetting agents,alcohols, fatty acids, and/or emulsifiers, particularly in liquidformulations. In some embodiments, the engineered PAL polypeptides areadministered to subjects in combination with other compounds used in thetreatment of PKU, including but not limited to KUVAN®tetrahydrobiopterin (BioMarin Pharmaceutical, Inc., Novato, Calif.),antacids (e.g., omeprazole, esomeprazole and other prazoles), as well asany other suitable compounds.

In some embodiments, the present invention provides engineered PALpolypeptides suitable for use in decreasing the concentration ofphenylalanine in fluids such as blood, cerebrospinal fluid, etc. Thedosage of engineered PAL polypeptide(s) administered to an animal dependupon the condition or disease, the general condition of the animal, andother factors known to those in the art. In some embodiments, thecompositions are intended for single or multiple administration to ananimal. In some embodiments, it is contemplated that the concentrationof engineered PAL polypeptide(s) in the composition(s) administered toan animal (e.g., a human with PKU) is sufficient to effectively treat,ameliorate and/or prevent disease (e.g., PKU and/or PKU-relatedconditions, diseases and/or symptoms). In some embodiments, theengineered PAL polypeptides are administered in combination with otherpharmaceutical and/or dietary compositions.

Industrial Compositions

It is contemplated that the engineered PAL polypeptides of the presentinvention will find use in industrial compositions. In some embodiments,the engineered PAL polypeptides are formulated for use in the foodand/or feed industries. In some embodiments, the engineered PALpolypeptides are formulated in granulated or pelleted products which aremixed with animal feed components such as additional enzymes (forexample, cellulases, laccases, and amylases). In some alternativeembodiments, the engineered PAL polypeptides are used in liquid animalfeed compositions (e.g., aqueous or oil based slurries). Thus, in someembodiments, the engineered PAL variants of the present invention aresufficiently thermotolerant and thermostable to withstand the treatmentused to produce pellets and other processed feed/foods.

The engineered PAL variants of the present invention also find use inthe production of phenylalanine and/or phenylalanine derivatives.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following non-limiting examples. Theexamples are provided for illustrative purposes only and are notintended to limit the scope of the present invention in any way.

EXPERIMENTAL

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and l (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);psi and PSI (pounds per square inch); ° C. (degrees Centigrade); RT andrt (room temperature); CDS (coding sequence); DNA (deoxyribonucleicacid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratoryE. coli strain, available from the Coli Genetic Stock Center [CGSC], NewHaven, Conn.); HTP (high throughput); HPLC (high pressure liquidchromatography); CFSE (carboxyfluorescein succinimidyl ester); IPTG(isopropyl β-D-1-thiogalactopyranoside); PES (polyethersulfone); PHE andphe (phenylalanine); BSA (bovine serum albumin); PBMC (peripheral bloodmononuclear cells); PKU (phenylketonuria); MHC (major histocompatibilitycomplex); HLA (human leukocyte antigen); HLA-DR (an MHC Class II cellsurface receptor encoded by the HLA complex on chromosome #6); FIOPC(fold improvements over positive control); LB (Luria broth); AthensResearch (Athens Research Technology, Athens, Ga.); ProSpec (ProSpecTany Technogene, East Brunswick, N.J.); Sigma-Aldrich (Sigma-Aldrich,St. Louis, Mo.); Ram Scientific (Ram Scientific, Inc., Yonkers, N.Y.);Pall Corp. (Pall, Corp., Pt. Washington, N.Y.); Millipore (Millipore,Corp., Billerica Mass.); Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); Molecular Devices (Molecular Devices, LLC,Sunnyvale, Calif.); Kuhner (Adolf Kuhner, AG, Basel, Switzerland);Cambridge Isotope Laboratories, (Cambridge Isotope Laboratories, Inc.,Tewksbury, Mass.); Applied Biosystems (Applied Biosystems, part of LifeTechnologies, Corp., Grand Island, N.Y.), Agilent (Agilent Technologies,Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo FisherScientific, Waltham, Mass.); Corning (Corning, Inc., Palo Alto, Calif.);Constant Systems (Constant Systems Ltd., Daventry, United Kingdom);Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo LifeSciences, Inc., Farmingdale, N.Y.); GE Healthcare (GE HealthcareBio-Sciences, Piscataway, N.J.); Harlan (Harlan Laboratories,Indianapolis, Ind.); AB Sciex (AB Sciex, Framingham, Mass.); and Bio-Rad(Bio-Rad Laboratories, Hercules, Calif.).

The following polynucleotide and polypeptide sequences find use in thepresent invention. In some cases (as shown below), the polynucleotidesequence is followed by the encoded polypeptide.

Polynucleotide Sequence of pET16b-AvPAL Expression Vector(SEQ ID NO: 1): (SEQ ID NO: 1)TCTCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCCTTAATGCAGACACGGCAGAATGTCCTGAACGGCCTGAACAATAACACCACCGGCTGCAATATCTGCACTAATACGTGCAATATGTTCATCCAGACCCTGTTCATTATCATTCCAAATATACGGACGATCTGAGGTCGGTTTCTGACCAACAACATGACGAACTGCGCTATACAGACGTTCGGTTGCCGGTGACAGACAGGCACGTGCATCATAATGACCGGTTTTTTTGTAGGTACGCAGATCAACTGCCTGAACACCAAACATCAGGGCAATGGCAACATAATTCTGAAAAATATCAACGCTACGACGTGCCAGGGTTGCGCTGGTATAACCCTGGCTGTTAATATTCTGGTTAAACTGTTCGGCATGGGTCGGAAAACGATCTGCAATACTATTACCATAAAAGGTCAGCAGCGGCATAATGCTATTACCGCAAATCTGCAGACCTTTCAGACCCATATTAACTTTACGTTCACGATTACCCAGCAGACTCGGAGGCAGACCATTGCTAAATTCCGGTGATGCCAGCAGTGCAATCTGAACATCCAGATGTTTTGCCAGCAGACCGATATAATAGCGCAGATGATCCATACCCATACCAACATACTGACCCAGAAAATTACCACCATGATAGCTTGCCTGATTATCAACATCAATCAGCGGGTTATCGGTAACGCTGTTAATCTCAATTTCGATTTGTTTGGCAATCTGGCTAATACCATCAACAATCGGACCCAGATACTGCGGCAGACAACGCAGGCTATAACGATCCTGGATCAGTTCATGATCACGATAATCATGTTTACCATCCAGTTCATCACGAACCAGCTGGCTATTGGCCAGCAGGCTAATCATCTGATCTGCTGCCCACAGCTGACCCGGATGCGGTTTGCTGTTATGGATAAACGGATGAAAGCTCTGATTTGTACCATTCAGTGCCTGAATATCCAGTGCATGAACACCCATTGCAATTGCGGTCAGAATCTGGGTATCATAAACACAATTTGCTGCAATACCGGTCATAACGCTGGTGCCATTCATCATTGCCAGACCTTCTTTCGGCAGCAGGGTCAGCGGACTCAGATTCAGCTGACGCAGTGCGGTCGGTGCGTCCATTTCTTTGCCATTAAAATCAACTTTAAAGCTCGGGTCCAGGCCAATCAGGCTACCGGTAATATAGCTCAGCGGAACCAGATCACCGCTGGCACCAATGCTACCAAATTCATAAACATACGGGGTAACACCGGCATTCAGAAAGATTTCCATGCGTTTAATCAGTTCCAGACGAATACCGCTTGCACCACGCATGTGGCTATTTGCACGCAGCAGCATTGCTGCACGAACATCTGCCAGCGGCAGTTTATTACCTGCACCGGTTTTCAGAAACCAAACCAGATTGGTCTGCAGTTCGCTTGCCTGTTCACGGCTAATTGCAACATTTGCCATACCACCAAAACCGCTGGTAACACCATAAATCGGTTCACCGCTTTCAACTGCATTATTGATATAATCACAGCTGGCCTGAATACCCTGCAGAATATCGGTATTATTGGTCAGGCTAACCAGGGTGCCATTACGGGCAACACGTGCAACATCATTGATGGTCAGTTTCTGATTACCAATAATCACATTTGCGCTGCTATTGCCGGTAAAGCTAAACTGCTGGCTGCTGGTTTTGCTCTGTGCCTGGCTCAGGGTTTTCATATGACGACCTTCGATATGGCCGCTGCTGTGATGATGATGATGATGATGATGATGATGGCCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATPolynucleotide Sequence of the AvPAL ORF (SEQ ID NO: 2): (SEQ ID NO: 2)ATGAAAACCCTGAGCCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGCAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCAATAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATCATGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGCTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGACCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATPolynucleotide Sequence of WT AvPAL (SEQ ID NO: 3): (SEQ ID NO: 3)ATGAAAACCCTGAGCCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGCAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCAATAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATCATGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGCTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGACCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATPolypeptide Sequence of WT AvPAL (SEQ ID NO: 4): (SEQ ID NO: 4)MKTLSQAQSKTSSQQFSFTNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVAISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVVDTILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLANSQLVRDELDGKHDYRDHELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLLGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQKPTSDRPYTWNDNEQGLDEHIARISADIAAGGVIVQAVQDI LPCLHPolynucleotide Sequence of AvPAL Variant No. 30 (SEQ ID NO: 5):(SEQ ID NO: 5)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGTACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGGACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATCATGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGTATCACCGGAATTTAACAATGGTCTGCCTGCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGTATCCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATPolypeptide Sequence of AvPAL Variant No. 30 (SEQ ID NO: 6):(SEQ ID NO: 6)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVVRVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQGLNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDHELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLVSPEFNNGLPASLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQYPSSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQ DILPCLHPolynucleotide Sequence of AvPAL Variant No. 22 (SEQ ID NO: 7):(SEQ ID NO: 7)ATGAAAACCCTGAGCCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGCAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCAATAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGCTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGACCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATPolypeptide Sequence of AvPAL Variant No. 22 (SEQ ID NO: 8):(SEQ ID NO: 8)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVS LTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVAISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLANSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLLGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQKPTSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDI LPCLHPolynucleotide Sequence of AvPAL Variant No. 36 (SEQ ID NO: 9):(SEQ ID NO: 9)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGTACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATPolypeptide Sequence of AvPAL Variant No. 36 (SEQ ID NO: 10):(SEQ ID NO: 10)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVVRVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQKPSSDRPYIVVNDNEQGLDEHIARISADIAAGGVIVQAVQDI LPCLHPolynucleotide Sequence of AvPAL Variant No. 42 (SEQ ID NO: 11):(SEQ ID NO: 11)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGTACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATAAAGATATTCTGCAGCGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAAAGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATATGGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTAAAAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGCCGCTGCATPolypeptide Sequence of AvPAL Variant No. 42 (SEQ ID NO: 12):(SEQ ID NO: 12)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVVRVARNGTLVSLTNNKDILQRIQASCDYINNAVEKGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYMDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGKKPSSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDILPPLHPolynucleotide Sequence of AvPAL Variant No. 43 (SEQ ID NO: 13):(SEQ ID NO: 13)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGTACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATAAAGATATTCTGCAGCGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATATGGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTAAAAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of AvPAL Variant No. 43 (SEQ ID NO: 14):(SEQ ID NO: 14)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVVRVARNGTLVSLTNNKDILQRIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYMDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGKKPSSDRPYIVVNDNEQGLDEHIARISADIAAGGVIVQAVQDILPNLHPolynucleotide Sequence of AvPAL Variant No. 1002 (SEQ ID NO: 15):(SEQ ID NO: 15)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCGCGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCATGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCGCGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTGGCCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTGGCCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of AvPAL Variant No. 1002 (SEQ ID NO: 16):(SEQ ID NO: 16)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMHGASGIRLELIKRAEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGGLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIGQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGQKPSSDRPYIWNDNEQGLDE HIARISADIAAGGVIVQAVQDILPNLHPolynucleotide Sequence of AvPAL Variant No. 1008 (SEQ ID NO: 17):(SEQ ID NO: 17)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCGCGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCATGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCGCGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTGGCCTGGCAAAACATCTGGATACCCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTGGCCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of AvPAL Variant No. 1008 (SEQ ID NO: 18):(SEQ ID NO: 18)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMHGASGIRLELIKRAEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGGLAKHLDTQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIGQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGQKPSSDRPYIVVNDNEQGLDE HIARISADIAAGGVIVQAVQDILPNLHPolynucleotide Sequence of AvPAL Variant No. 1009 (SEQ ID NO: 19):(SEQ ID NO: 19)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCGCGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCATGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCGCGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATGAAATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTGGCCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of AvPAL Variant No. 1009 (SEQ ID NO: 20):(SEQ ID NO: 20)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMHGASGIRLELIKRAEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYEIGLLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIGQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGQKPSSDRPYIVVNDNEQGLDEHIARISADIAAGGVIVQAV QDILPNLHPolynucleotide Sequence of AvPAL Variant No. 1010 (SEQ ID NO: 21):(SEQ ID NO: 21)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCGCGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCATGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCGCGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATGAAATCGGTCTGCTGGCAAAACATCTGGATACCCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTGGCCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of AvPAL Variant No. 1010 (SEQ ID NO: 22):(SEQ ID NO: 22)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMHGASGIRLELIKRAEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYEIGLLAKHLDTQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIGQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGQKPSSDRPYIVVNDNEQGLDE HIARISADIAAGGVIVQAVQDILPNLHPolynucleotide Sequence of AvPAL Variant No. 1084 (SEQ ID NO: 23):(SEQ ID NO: 23)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCCATACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGTACGTGTTGCCCGTAATGGCACCGCGGTTAGCCTGACCAATAATAAAGATATTCTGCAGCGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAAAGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCAGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATATGGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTAAAAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGCCGCTGCATPolypeptide Sequence of AvPAL Variant No. 1084 (SEQ ID NO: 24):(SEQ ID NO: 24)MKTLSQAQSKTSSQQFSHTGNSSANVIIGNQKLTINDVVRVARNGTAVSLTNNKDILQRIQASCDYINNAVEKGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLQPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYMDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGKKPSSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDILPPLHPolynucleotide Sequence of AvPAL Variant No. 967 (SEQ ID NO: 25):(SEQ ID NO: 25)ATGAAAACCCTGAGTCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCGCGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGTAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCGCGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCGGTAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATGGTGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTGGCCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGGTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTGGCCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCCAGCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGAGCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGAACCTGCATPolypeptide Sequence of Variant No. 967 (SEQ ID NO: 26): (SEQ ID NO: 26)MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVVISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRAEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLAGSQLVRDELDGKHDYRDGELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGGLAKHLDVQIALLASPEFSNGLPPSLVGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIGQNYVAIALMFGVQAVDLRTYKKTGHYDARAQLSPATERLYSAVRHVVGQKPSSDRPYIWNDNEQGLDE HIARISADIAAGGVIVQAVQDILPNLHExpression vector pCK100900i: (SEQ ID NO: 27)TGGCCACCATCACCATCACCATTAGGGAAGAGCAGATGGGCAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTTGAATTCTACGTAAAAAGCAGCCGATACATCGGCTGCTTTTTTTTTGNNNGAGGTTCCAACTTGTGGTATAATGAAATAAGATCACTCCGGAGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAGGAACTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAACTGCAGGAGCTCAAACAGCAGCCTGTATTCAGGCTGCTTTTTTCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGAACCACCGCTGGTAGCGGTGGTTTTTTTAGGCCTATGGCCTTTTTTTTTTNTGNNAAACCTTTCGCGGTATGGNATNANAGCGCCCGGAAGAGAGTCAATTAAGAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGGTACCCGATAAAAGCGGCTTCCTGACAGGAGGCCGTTTTGTTTCTCGAGTTAATTAAGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAATCTAGAGGCCAGCCTGGCCATAAGGAGATATACATATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGAAAACCCTGAGCCAGGCACAGAGCAAAACCAGCAGCCAGCAGTTTAGCTTTACCGGCAATAGCAGCGCAAATGTGATTATTGGTAATCAGAAACTGACCATCAATGATGTTGCACGTGTTGCCCGTAATGGCACCCTGGTTAGCCTGACCAATAATACCGATATTCTGCAGGGTATTCAGGCCAGCTGTGATTATATCAATAATGCAGTTGAAAGCGGTGAACCGATTTATGGTGTTACCAGCGGTTTTGGTGGTATGGCAAATGTTGCAATTAGCCGTGAACAGGCAAGCGAACTGCAGACCAATCTGGTTTGGTTTCTGAAAACCGGTGCAGGTAATAAACTGCCGCTGGCAGATGTTCGTGCAGCAATGCTGCTGCGTGCAAATAGCCACATGCGTGGTGCAAGCGGTATTCGTCTGGAACTGATTAAACGCATGGAAATCTTTCTGAATGCCGGTGTTACCCCGTATGTTTATGAATTTGGTAGCATTGGTGCCAGCGGTGATCTGGTTCCGCTGAGCTATATTACCGGTAGCCTGATTGGCCTGGACCCGAGCTTTAAAGTTGATTTTAATGGCAAAGAAATGGACGCACCGACCGCACTGCGTCAGCTGAATCTGAGTCCGCTGACCCTGCTGCCGAAAGAAGGTCTGGCAATGATGAATGGCACCAGCGTTATGACCGGTATTGCAGCAAATTGTGTTTATGATACCCAGATTCTGACCGCAATTGCAATGGGTGTTCATGCACTGGATATTCAGGCACTGAATGGTACAAATCAGAGCTTTCATCCGTTTATCCATAACAGCAAACCGCATCCGGGTCAGCTGTGGGCAGCAGATCAGATGATTAGCCTGCTGGCCAATAGCCAGCTGGTTCGTGATGAACTGGATGGTAAACATGATTATCGTGATCATGAACTGATCCAGGATCGTTATAGCCTGCGTTGTCTGCCGCAGTATCTGGGTCCGATTGTTGATGGTATTAGCCAGATTGCCAAACAAATCGAAATTGAGATTAACAGCGTTACCGATAACCCGCTGATTGATGTTGATAATCAGGCAAGCTATCATGGTGGTAATTTTCTGGGTCAGTATGTTGGTATGGGTATGGATCATCTGCGCTATTATATCGGTCTGCTGGCAAAACATCTGGATGTTCAGATTGCACTGCTGGCATCACCGGAATTTAGCAATGGTCTGCCTCCGAGTCTGCTGGGTAATCGTGAACGTAAAGTTAATATGGGTCTGAAAGGTCTGCAGATTTGCGGTAATAGCATTATGCCGCTGCTGACCTTTTATGGTAATAGTATTGCAGATCGTTTTCCGACCCATGCCGAACAGTTTAACCAGAATATTAACAGCCAGGGTTATACCAGCGCAACCCTGGCACGTCGTAGCGTTGATATTTTTCAGAATTATGTTGCCATTGCCCTGATGTTTGGTGTTCAGGCAGTTGATCTGCGTACCTACAAAAAAACCGGTCATTATGATGCACGTGCCTGTCTGTCACCGGCAACCGAACGTCTGTATAGCGCAGTTCGTCATGTTGTTGGTCAGAAACCGACCTCAGATCGTCCGTATATTTGGAATGATAATGAACAGGGTCTGGATGAACATATTGCACGTATTAGTGCAGATATTGCAGCCGGTGGTGTTATTGTTCAGGCCGTTCAGGACATTCTGCCGTGTCTGCATTAAGGCCAAAC

Example 1 PAL Gene Acquisition and Construction of Expression Vectors

Anabaena variabilis phenylalanine ammonia lyase (AvPAL) plasmid DNA wasobtained and a synthetic gene encoding AvPAL was codon optimized forexpression in E. coli and cloned into the E. coli expression vectorpET16b to provide pET16b-AvPAL (SEQ ID NO:1). The AvPAL open readingframe (SEQ ID NO:2) was amplified by PCR using the oligonucleotides:PAL-pCK-F and PAL-pCK-R and subcloned into the expression vectorpCK100900i (SEQ ID NO: 27).

Primer Sequence 5' to 3' SEQ ID NO: PAL-CTAGAGGCCAGCCTGGCCATAAGGAGATATACAT SEQ ID NO: 28 pCK-FATGAAAACCCTGAGCCAGGCAC PAL- GATGGTGATGGTGGCCAGTTTGGCCTTAATGCAGSEQ ID NO: 29 pCK-R ACACGGCAGAATG

This plasmid construct was transformed into an E. coli strain derivedfrom W3110. Directed evolution techniques generally known by thoseskilled in the art were used to generate libraries of gene variants fromthis plasmid construct (See e.g., U.S. Pat. No. 8,383,346 andWO2010/144103).

Example 2 High-Throughput (HTP) Growth and Assays

In this Example, methods used for HTP growth and various assays used totest the variant PALs are described.

High-Throughput (HTP) Growth of PAL and PAL Variants

Transformed E. coli cells were selected by plating onto LB agar platescontaining 1% glucose and 30 μg/ml chloramphenicol. After overnightincubation at 37° C., colonies were placed into NUNC™(Thermo-Scientific) the wells of 96-well shallow flat bottom platesfilled with 180 μl/well LB supplemented with 1% glucose and 30 μg/mlchloramphenicol. The cultures were allowed to grow overnight for 18-20hours in a shaker (200 rpm, 30° C., and 85% relative humidity; Kuhner).Overnight growth samples (20 μL) were transferred into Costar 96-welldeep plates filled with 3804 of Terrific Broth supplemented with 30μg/ml chloramphenicol. The plates were incubated for 135 minutes in ashaker (250 rpm, 30° C., and 85% relative humidity; Kuhner). The cellswere then induced with 40 μL of 10 mM IPTG in sterile water andincubated overnight for 20-24 hours in a shaker (250 rpm, 30° C., and85% relative humidity; Kuhner). Two replicate cultures were combined,the cells were pelleted (4000 rpm×20 min), the supernatants werediscarded, and the cells were frozen at −80° C. prior to analysis.

Lysis of HTP Pellets

First, 500 μL of lysis buffer (20 mM Tris pH 7.5, 1 mM MgSO₄, 1 mg/mllysozyme, and 0.5 mg/ml polymyxin B sulfate) were added to the cellpellets. The mixture was agitated for 1.5 h at room temperature, andpelleted (4000 rpm×5 min) prior to use of the clarified lysates in thevarious HTP assays described herein. Analysis of these lysates bySDS-PAGE revealed the presence of an overexpressed protein at anapparent MW of ˜60 kDa, consistent with the expected MW of PAL.

Analysis of Clarified Lysates

PAL variant activity was determined by measuring the formation ofcinnamic acid as determined by the change in absorbance at 290 nm overtime. For this assay, 100 μL of either 200 mM Tris/50 mM phenylalanine,pH 7.5, or 200 mM sodium phosphate/50 mM phenylalanine pH 7.0, 80 μL ofwater, and 20 μL of clarified lysate were added to the wells of apoly-acrylate 96-well plate (Costar #3635, Corning). The reactions weremixed briefly and the activity was determined by tracking the absorbanceat 290 nm over time (every 12-20 s over 5-20 min) using a SpectraMax®Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbance microplatereader.

HTP-Analysis of Clarified Lysates Pretreated with Protease

PAL variants were challenged with chymotrypsin and trypsin to simulatethe environment of the lower intestine. First, 30 μL of protease mix(0.01-100 mg/ml chymotrypsin (C4129 Sigma Aldrich), 0.01-100 mg/mltrypsin (T7409 Sigma Aldrich), 1 mM CaCl₂, and 1 mM HCl), 0-304 of 20 mMsodium taurocholate in 500 mM sodium phosphate pH 7.0, and 90-120 μL ofclarified lysate were added to the wells of a 96-well round bottom plate(Costar #3798, Corning). The plates were sealed and incubated at 37° C.,400 rpm, 1″ throw for 1 h prior to analysis. For the assay, 100 μL ofeither 200 mM Tris/50 mM phenylalanine pH 7.5 or 200 mM sodiumphosphate/50 mM phenylalanine pH 7.0 and 100 μL of the protease treatedlysate were added to the wells of a poly-acrylate 96-well plate (Costar#3635, Corning). The reactions were mixed briefly and the activity wasdetermined by tracking the absorbance at 290 nm over time (every 12-20 sover 5-20 min) using a SpectraMax® Plus³⁸⁴ or a SpectraMax® 190(Molecular Devices) absorbance microplate reader. The results areprovided in the following Tables.

HTP-Analysis of Clarified Lysates Pretreated with Acid

In this assay, PAL variants were challenged under acidic conditions, inorder to simulate the environment of the stomach. First, 20 μL of 1Msodium citrate (pH 3.7-4.5) and 30 μL of water or 50 μL of 400 mM sodiumcitrate pH 3.7-4.8, and 50 uL of clarified lysate were added to thewells of a 96-well round bottom plate (Costar #3798, Corning). The platewas sealed and incubated at 37° C., 400 rpm, 1″ throw for 1 h prior toanalysis. For the assay, 100 μL of either 200 mM Tris, 50 mMphenylalanine pH 7.5 and 80 μL 1M Tris pH 7.5 or 200 mM sodiumphosphate/50 mM phenylalanine pH 7.0 and 80 μL of 1.0 M sodium phosphatepH7.0, and 20 μL of the acid-treated lysate were added to the wells of apoly-acrylate 96-well plate (Costar #3635, Corning). The reactions weremixed briefly, and the activity was determined by tracking theabsorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The results are provided in the following Tables.

HTP Analysis of Clarified Lysates Pretreated with Pepsin

In additional assays, PAL variants are challenged with acidic conditionsand pepsin to further test the variants under conditions that mimic thegastric environment. First, 50 μL of 0.01-100 mg/ml pepsin in 400 mMsodium citrate pH 1.5-4, and 50 μL of clarified lysate are added to thewells of a 96-well round bottom plate (Costar #3798, Corning). The plateis sealed and incubated at 37° C., 400 rpm, 1″ throw for 1-12 h prior toanalysis. For the assay, 100 μL of either 200 mM Tris/50 mMphenylalanine pH 7.5 and 80 μL 1M Tris pH 7.5 or 200 mM sodiumphosphate/50 mM phenylalanine pH 7.0, and 20 μL of acid-treated lysateare added to the wells of a poly-acrylate 96-well plate (Costar #3635,Corning). The reactions are mixed briefly, and the activity isdetermined by tracking the absorbance at 290 nm over time (every 12-20 sover 5-20 min) using a SpectraMax® Plus³⁸⁴ or a SpectraMax® 190(Molecular Devices) absorbance microplate reader.

TABLE 2-1 Relative Activity of PAL Variants When Unchallenged (U-C),Protease-Challenged (P-C), and Acid-Challenged (A-C). Variant Amino AcidDifferences # U-C P-C A-C Relative to SEQ ID NO: 4 1 + + +A39V/A91V/Y158H/S180A/N290G/ A394V/S399N/N474Q/K522Y/T524S 2 + + ++A39V/A91V/Y158H/A256G/A394V/ P404A/N474Q 3 + ++ ++A39V/A91V/S180A/A394V/K522F/ T524S 4 + + ++ A39V/A91V/Y158H/T243I/A256G/S399N/P404A/L407V/N474Q 5 + + ++ E308Q 6 + ++ + N400K 7 + + + L364Q8 + + ++ A256G/N290G/P404A/L407V/N474Q/ K522F 9 + + ++Y158H/S180A/A394V/T463N/N474Q/ T524S 10 + + ++A39V/A91V/Y158H/S180A/K195E/ A256G/S399N/L407V/Y459F/T463N/ K522N/T524S11 + + ++ A39V/A91V/Y158H/S180A/K195E/ A256G/N290G/S399N/Y459F/T463N12 + ++ ++ A39V/A91V/Y158H/S180A/K195E/ T243I/A394V 13 + ++ ++A39V/A91V/Y158H/K195E/T243I/ A256G/A394V/S399N/N474Q/ K522Y/T524S 14 +++ ++ A39V/A91V/Y158H/S180A/K195E/ I245L/S399N/L407V/Y459F/T463N/N474Q/T524S 15 + ++ + H307M 16 + + ++ A39V/A91V/Y158H/S180A/T243L/A394V/S399N/T463N/K522F 17 + + ++ A91V/N474Q 18 ++ + ++ S180A/K195E 19++ + ++ A91V/Y158H/T243I/A256G/N290G/ S399N/L407V/Y459F/T463N/N474Q/K522N/T524S 20 ++ ++ ++ A39V/Y158H/S180A/S399N 21 ++ ++ ++ A91V/N270K 22++ +++ ++ H307G 23 ++ ++ + N453G 24 ++ ++ ++ H307Q 25 ++ ++ + L257W 26++ + +++ A91V/Y158H/I245L/A256G/S399N/ Y459F/T463N/K522Y/T524S 27 ++++ + F443H 28 ++ ++ ++ A91V/Y158H/S180A 29 ++ ++ +A91Y/Y158H/K195E/S399N 30 ++ ++ +++ A39V/A91V/A256G/N290G/A394V/S399N/P404A/L407V/K522Y/T524S 31 ++ ++ + L257A 32 ++ + +++Y158H/K195E/T243L/A256G/A394V/ S399N/L407V/N474Q/K522F/T524S 33 ++ ++L349M 34 +++ +++ + I326F 35 +++ +++ +++ T460G/P528L 1. Relative activitywas calculated as activity of the variant/activity of SEQ ID NO: 4(encoded by SEQ ID NO: 3). 2. Variant # 22 has the polynucleotidesequence of SEQ ID NO: 7 and polypeptide sequence of SEQ ID NO: 8, andVariant # 30 has polynucleotide sequence of SEQ ID NO: 5 and polypeptidesequence of SEQ ID NO: 6. 3. + = 0.1 to 1.0 relative activity overwild-type AvPAL; ++ = >1.0 to 2.0 relative activity over wild-typeAvPAL; and +++ = >2.0 relative activity over wild-type AvPAL.

TABLE 2-2 Relative Activity of PAL Variants Unchallenged (U-C),Protease-Challenged (P-C), and Acid-Challenged (A-C). Amino AcidDifferences - Variant Relative to Wild-Type # U-C P-C A-C AvPAL (SEQ IDNO: 4) 36 ++ +++ ++ A39V/A91V/N290G/H307G/L407V/ T524S 37 ++ +++ ++A39V/A91V/N290G/H307G/L407V 38 + ++ + A39V/A91V/A256G/N290G/H307G/S399N/P404A/L407V/K522Y/T524S 39 ++ + ++ A39V/A91V/A256G/N290G/S399N/P404A/L407V/K522Y 40 + ++ + A39V/A256G/N290G/H307G/P404A/ L407V 41 +++ + A39V/A91V/A256G/N290G/H307G/ P404A/L407V/T524S 1. Relative activitywas calculated as activity of the variant/activity of Variant No. 30. 2.Variant # 36 has polynucleotide sequence of SEQ ID NO: 9 and polypeptidesequence of SEQ ID NO: 10. 3. + = >1.0 to 3.0 relative activity overVariant 30; ++ = >3.0 to 10 relative activity over Variant 30; and +++= >10 to 35 relative activity over Variant 30.

TABLE 2-3 Relative Activity of PAL Variants Unchallenged (U-C),Protease-Challenged (P-C), and Acid-Challenged (A-C). Variant # U-C A-CP-C Mutations (Relative to Variant No. 36) 42 + + +T54K/G59R/S73K/R305M/ C503Q/Q521K/C565P 43 + +++ ++ T54K/G59R/R305M/C503Q/Q521K/C565N 44 ++ +++ ++ G59R/C503Q 45 ++ +++ ++K32P/G59R/S73K/Q240W/C503Q/C565N 46 + + + K32P/G59R/S73K/Q240W/C565P47 + +++ ++ K32P/T54K/S73K/R305M/ C503Q/Q521K/C565N 48 +++ +++ ++Y304H/D353A 49 ++ +++ +++ S73K/D353A 50 + + ++ A112C/S546R 51 ++ + ++S73K/Q240W/Y304H 52 + + ++ R134Q/Q240W/Y304H/D353A/E509L 1. Relativeactivity was calculated as activity of variant/activity of Variant No36. 2. Variant # 42 has the polynucleotide sequence of SEQ ID NO: 11 anda polypeptide sequence of SEQ ID NO: 12. Variant # 43 has thepolynucleotide sequence of SEQ ID NO: 13 and a polypeptide sequence ofSEQ ID NO: 14. 3. + = >0.5 to 1.5 relative activity over Variant 36; ++= >1.5 to 3 relative activity over Variant 36; and +++ = >3 to 10relative activity over Variant 36.

TABLE 2-4 Relative Activity of PAL Variants Unchallenged (U-C),Protease-Challenged (P-C), and Acid-Challenged (A-C). Variant # U-C P-CA-C Mutations (Relative to Variant: 30) 53 + + + D303R 54 + + ++ E308A55 + + + E308D 56 ++ ++ + G256A 57 + ++ + H307A 58 + +++ + H307D 59 +++ + H307E 60 + ++ + H307F 61 + +++ + H307G 62 + + + H307I 63 + + +H307L 64 ++ +++ ++ H307M 65 + ++ + H307N 66 ++ ++ ++ H307R 67 + + +H307Y 68 + +++ + R305L 69 + +++ + R305M 70 + +++ + R305Q 71 ++ ++ +V91A/G256A 72 + ++ + Y304H 73 + + + Y304W 74 + + + C503K 75 + + + C503Q76 + + + C565A 77 + + + C565G 78 + + + C565I 79 + + + C565K 80 + + +C565L 81 + ++ + C565N 82 + + + C565P 83 + + + C565T 1. Relative activitywas calculated as activity of variant/activity of Variant No. 30(Variants 53-73) or wild-type AvPAL of SEQ ID NO: 4 (encoded by SEQ IDNO: 3) (Variants 74-83.) 2. + = >0.5 to 1.5 relative activity overVariant 30 or wild-type AvPAL; ++ = >1.5 to 3 relative activity overVariant 30 or wild-type AvPAL; and +++ = >3 to 10 relative activity overVariant 30 or wild-type AvPAL.

TABLE 2-5 Relative Activity of PAL Variants Unchallenged (U-C),Protease-Challenged (P-C), and Acid-Challenged (A-C). Variant # U-C P-CA-C Mutations Relative to Variant 42 42 + + + 1011 + + +R59G/R134Q/Q240R/K521Q/P564L 1012 + + ++ R59G/P564M 1013 + + +R59G/Q240W/E509L/K521Q/P564M 1014 ++ ++ + Q240W/Y304H/D353N/E509L/K521S/P564L 1015 + ++ + R59G/R134Q/Q240R/K521Q/P565N 1016 + +++ +Y304H/D353A/E509L 1017 + ++ + R59G/R134Q/Y304H/D353N/K521Q 1018 + +++ +R59G/R134Q/Q240R/K521S 1019 + + + R134Q/Y304H/D353A/K521S/P565N1020 + + + R59G/R134Q/Q240R/Y304H/ D353A/K521Q/P564M 1021 ++ +++ ++Q240W/D353A/E509L/K521Q 1022 + ++ + R134Q/Q240W/E509L/K521S 1023 ++ ++++ R134Q/Q240R/D353A/K521Q/P564G 1024 + + + R134Q/K521Q 1025 + +++ +K521Q 1026 + +++ + R134Q/D353A/K521S/P564M 1027 + +++ +R59G/R134Q/Q240W/D353A 1028 ++ ++ + R59G/K521S 1029 + ++ + R59G/D353A1030 + + + Q240R/D353A/K521S 1031 + + + R59G/D353A/E509L/P564L1032 + + + R59G/D353A/K521Q 1033 + + + R59G/R134Q/D353A/E509L/K521Q1034 + + + Q240R/Y304H/E509L 1035 + + + R59G/Q240R/D353A/E509L1036 + + + R59G/R134Q/E509L 1037 + + + Q240W/Y304H/E509L/K521S1038 + + + Q240W/D353A/E509L/ K521S/P564L/P565N 1039 + + +R59G/Q240R/D353A/P564M/P565N 1040 + + + R134Q/D353A/K521S 1041 + + +R134Q/Q240W/Y304H/D353A/P564L 1042 + + + Q240R/E509L/P565N 1043 + + +Q240R/K521S/P564M/P565N 1044 + + + R134Q/D353A/P564L 1045 + + +R59G/R134Q 1046 + + + R59G/Q240R 1047 + + + Y304H/E509L 1048 + + +R134Q/Q240R/D353A/ E509L/K521Q/P564L 1049 + + + Q240W/E509L/P564L/P565N1050 + + + R59G/E509L/P564L 1051 + + + R134Q/D353A/E509L/P564L1052 + + + R59G/D353A/P565N 1053 + + + Q240R/E509L 1054 + + + Q240R1055 + + + D353A/K521Q 1056 + + + Q240W/D353A/E509L 1057 + + +F18H/L47A/L214Q/E540D 1058 + + + F18H/L47A/L214Q/E308K/F450Y/S546R1059 + + + F18H/L47A/F450Y/P528L/S546R 1060 + + + L214Q/E308Q/T460G1061 + + + F18H/F450Y/E540D 1062 + + + P528L/S546R 1063 + + +F18H/L214Q/F450Y 1064 + + + E308Q/F450Y/R467G 1065 + + + F18H/E540D1066 + + + L47A/L214Q/S546R 1067 + + + F18H/L214Q/E308Q 1068 + + +L47A/L214Q/E308Q 1069 + + + F18H/L47E/L214Q/R467G/E540D/S546R 1070 + + +L47A/F450Y 1071 + + + L47A/L214Q 1072 + + + F18H/L47A/L214Q/S546R1073 + + + F18H/L214Q 1074 + + + L214Q/F450Y/P528L 1075 + + +F18H/L214Q/E308Q/F450Y/R467G 1076 + + + L47A/L214Q/E540D 1077 + + +L214Q/E540D 1078 + + + F18H/L47A/E308Q/S546R 1079 + + + F18H 1080 + + +F18H/L47A/F450Y/S546R 1081 + + + F18H/L47A/L214Q/F450Y 1082 + + +F18H/L47A 1083 + + + L47A 1084 + + + F18H/L47A/L214Q 1085 + + +L214Q/S546R 1086 + + + F18H/L214Q/R467G/S546R 1087 + + +F18H/L47A/L214Q/E308Q 1088 + + + F18H/L214Q/T460G 1089 + + +F18H/L47A/F450Y 1090 + + + F18H/L214Q/E540D 1. Relative activity wascalculated as activity of variant/activity of Variant No. 42 2. − < 0.5relative activity over Variant No. 42; + = >0.5 to 1.5 relative activityover Variant No. 42; and ++ = >1.5 to 3 relative activity over VariantNo. 42.

Example 3 Assays to Determine Protein Aggregation of PAL Variants

The propensity of the PAL variants to aggregate is determined using theProteoStat® Protein Aggregation Assay kit (Enzo), according to themanufacturer's instructions. Briefly, purified PAL at 0-100 μM is mixedwith ProteoStat® detection reagent (1:2000) and analyzed via flowcytometry. Samples are assessed for fluorescence, consistent with theProteoStat® Aggregation Standards, as known in the art (See e.g.,Bershtein et al., Mol. Cell, 133-144 [2013]).

Example 4 Lyophilized Lysates from Shake Flask (SF) Cultures

Selected HTP cultures grown as described above were plated onto LB-agarplates with 1% glucose and 30 μg/ml chloramphenicol and grown overnightat 37° C. A single colony from each culture was transferred to 50 ml ofLB with 1% glucose and 30 μg/ml chloramphenicol. The cultures were grownfor 18 h at 30° C., 250 rpm, and subcultured at a dilution ofapproximately 1:10 into 250 ml of Terrific Broth with 30 μg/ml ofchloramphenicol, to a final OD600 of 0.2. The cultures were incubatedfor 135 minutes at 30° C., 250 rpm, to an OD600 of 0.6 and induced with1 mM of IPTG. The induced cultures were incubated for 20 h at 30° C.,250 rpm. Following this incubation period, the cultures were centrifuged4000 rpm×10 mM. The supernatant was discarded, and the pellets wereresuspended in 30 ml of 50 mM sodium phosphate pH 7.5. The cells werepelleted (4000 rpm×10 min), resuspended in 12 ml of 50 mM sodiumphosphate pH 7.5, and lysed using a One Shot Cell Disruption system(Constant Systems) at 17,000 psi. Lysate was pelleted (10,000 rpm×30min) and the supernatant was frozen and lyophilized to generate anenzyme-containing powder.

Purification of PAL from Shake Flask Cultures

PAL Variant No. 42 was grown in shake flask cultures to saturation, asdescribed above. Saturated cultures were pelleted by centrifugation(4000 rpm×20 min) and the cell pellets were stored at −80° C. prior topurification. The cell pellets were thawed at room temperature andresuspended in 25 mM Tris, pH 8 with 130 mM NaCl at 5 mL of buffer/g ofcells. The sample slurry was lysed using a microfluidizer with apressure setting of 110 psi. The resulting lysate was clarified bycentrifugation at 10,000 rpm for 1 hour, followed by filtration througha 0.2 μm PES filter (Millipore).

After filtration, the resulting lysate was heated at 70-85° C. for 1.5-2hours in the presence or absence of 10 mM Phe. The lysate was removedfrom the heat and clarified by centrifugation at 10,000 rpm at 4° C. for1 hour. The supernatant containing soluble PAL was then filtered througha 0.2 μm PES filter prior to loading onto a chromatography column.

The heat-treated, filtered lysate, containing 80-100 mg of totalprotein, was diluted two-fold using 25 mM Tris, pH 8 with 1.2 M ammoniumsulfate. The sample was loaded on to a HiPrep 16/10 Phenyl FF (hi sub)column (GE Healthcare) pre-equilibrated with the 25 mM Tris, pH 8 with0.6M ammonium sulfate. Following sample loading, the column was washedwith three column volumes of the same buffer, followed by a lineargradient of 0.6 M-0 M ammonium sulfate in 25 mM Tris, pH 8 for onecolumn volume. Tightly-bound PAL was eluted off the column using anisocratic elution with 25 mM Tris, pH 8 for three column volumes.Fractions containing active and pure PAL were pooled.

The purified PAL from the phenyl column was buffer-exchanged into 0.5 MTris, pH 8.5 and concentrated. The concentrated PAL was analyzed bySDS-PAGE and found to be present in a band at ˜60 kDa. The purified PALsamples were filtered using a 0.45 μm PES filter and were stored at −80°C. until ready for use.

Example 5 Characterization of Purified PAL and PAL Variants

In this Example, assays conducted to characterize wild-type and variantPALs are described.

Tolerance to Acidic pH:

Lyophilized powders containing PAL variants were dissolved at 2 g/L in20 mM sodium phosphate pH 7.0. Then, 50 μL of the enzyme solutions weremixed with 50 μL of 400 mM citric acid (pH 4.0-5.2) or 100 mM sodiumphosphate and reactions were incubated at 37° C. for 1 h at 400 rpm (1″throw). Then, 20 μL of the solution were briefly mixed with 80 μL of 1Msodium phosphate pH 7.0 and 100 μL of 200 mM Tris/50 mM phenylalanine pH7.5. The enzymatic activity under acidic conditions was determined bytracking the absorbance at 290 nm over time (every 12-20 s over 5-20 mM)using a SpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices)absorbance microplate reader. The results are shown FIG. 2. As indicatedin FIG. 2, Variant Nos. 30 and 36 maintained more activity after beingincubated at pHs approximately 4 to 4.8, compared to the wild-type PAL.

Determination of K_(M):

To evaluate whether the mutations in the variant PALs had altered theaffinity of the PAL variants for phenylalanine, the Michaelis constantfor the wild-type enzyme and Variant 36 were determined. First, 100 μLof 15 μg/ml PAL in 100 mM Tris pH 8.0, and 100 μL of 0-32 mMphenylalanine in 100 mM Tris, pH 8.0, were added to the wells of apoly-acrylate 96-well plate (Costar #3625, Corning). The reaction wasmixed briefly and initial rates were determined by tracking theabsorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The K_(M) for each tested PAL variant was determinedby fitting the data to a Lineweaver-Burke plot, as known in the art. Theresults are shown in FIG. 3. As shown, the K_(M) was 74 μM for thewild-type enzyme and 60 μM for Variant 36.

Amino Acid Specificity:

Some phenylalanine ammonia lyases demonstrate activity against tyrosineand/or histidine in addition to phenylalanine. To evaluate if themutations present in the PAL variants had altered the specificity of thePAL variants for phenylalanine, the activities of wild-type enzyme andVariant 36 on these three amino acids were assessed. First, 100 μL of 5g/L of PAL-containing lyophilized powder in 10 mM sodium phosphate pH7.0, and 100 μL of 50 mM phenylalanine or histidine or 2.5 mM tyrosinein 200 mM sodium phosphate pH 7.5 were added to the wells of apoly-acrylate 96-well plate (Costar #3635, Corning). The solutions weremixed briefly and initial rates were determined by tracking theabsorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The results are shown in FIG. 4. As indicated, nodetectable activity was observed for either the WT enzyme or Variant No.36 with either histidine or tyrosine, indicating that these enzymes arespecific for phenylalanine.

Resistance to Porcine and Bovine Proteases:

PAL variant samples prepared as described in Example 4, were dissolvedat 2 g/L in 100 mM sodium phosphate pH 7.0. Porcine trypsin and bovinechymotrypsin (100 mg each) were dissolved in 2 ml of 100 mM sodiumphosphate pH 7.0, and serially diluted 2-fold eleven times in 100 mMsodium phosphate. Then, 80 μL of the PAL variant enzyme solutions weremixed with 20 μL of the trypsin and chymotrypsin solution. The reactionmixtures were incubated at 37° C. for 1 h at 400 rpm (1″ throw). Then,20 μL of the reaction was mixed with 80 μL of water and 100 μL of 100 mMsodium phosphate, 50 mM phenylalanine pH 7.0. Each solution was mixedbriefly, and the activity was determined by tracking the absorbance at290 nm over time (every 12-20 s over 5-20 min) using a SpectraMax®Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbance microplatereader. The results are shown in FIG. 2. As indicated in this Figure,all of the tested variants showed improved protease resistance, ascompared to the wild-type PAL, with Variant No. 36 being the most stabletowards proteolysis.

Resistance to Human Proteases:

As described above, some evolved PAL variants were screened againstporcine trypsin and bovine chymotrypsin to assess their resistance toproteolysis by enzymes present in the gastrointestinal tract. Some ofthe evolved PAL variants were also tested using human enzymes, toconfirm that they are resistant to the human homologues of the porcineor bovine enzymes. In these assays, lyophilized powders of WT PAL andVariant No. 36 (2.4 g/L in 100 mM sodium phosphate, pH 7.0) wereincubated with human chymotrypsin (Athens Research) 0-80 BTEE units/mlor human trypsin (ProSpec) (0-10,000 BAEE units/ml) at 37° C. for 2 h.Then, 100 μL of the mixtures were added to the wells of a poly-acrylate96-well plate (Costar #3635, Corning), followed by the addition of 100μL of 50 mM phenylalanine, 200 mM sodium phosphate pH 7.0. The solutionwas mixed briefly and initial rates were determined by tracking theabsorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The results are shown in FIG. 5. As shown in FIG. 5,Variant No. 36 was more stable than the wild type PAL enzyme.

Resistance to Crude Pancreatic Extract:

The evolved PAL variants were also tested to determine their resistanceto pancreatic enzymes. Lyophilized powders of WT PAL, Variant No. 36,Variant No. 42, and Variant No. 43 lyophilized powders (prepared asdescribed in Example 4; 12 g/L in 50 mM potassium phosphate pH 6.8) weremixed 1:1 with porcine pancreatin (4× Sigma-Aldrich, St. Louis, Mo.),and incubated at 37° C. with shaking (400 rpm, 1″ throw) for up to 23 h.At the indicated time points, a 10 μL aliquot of the reactions was addedto 190 μL of 50 mM phenylalanine, 190 mM sodium phosphate pH 7.0, in thewells of a poly-acrylate 96-well plate (Costar #3635, Corning). Thereaction was mixed briefly and initial rates were determined by trackingthe absorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The results are shown in FIG. 6. As shown in FIG. 6,Variant No. 36, Variant No. 42, and Variant No. 43 all showedsignificant stability under these assay conditions, as compared to thewild-type PAL enzyme.

Impact of Intestinal Detergents:

The evolved PAL variants were also tested for their susceptibility toproteolysis in the presence or absence of intestinal bile acids andfatty acids, to assess whether these acids impact their stability.Lyophilized powders containing Variant No. 36 (prepared as described inExample 4) were dissolved at 50 μg/ml in 0-16 mM sodium taurocholate,100 mM sodium phosphate, pH 7.0. Porcine trypsin and bovine chymotrypsin(80 mg each) were dissolved in 2 ml of 100 mM sodium phosphate pH 7.0,and serially diluted 2-fold eleven times in 100 mM sodium phosphate. Forthe assay, 50 μL of the PAL solutions were mixed with 50 μL of theprotease solution. The mixtures were incubated at 37° C. for 1 h at 400rpm (1″ throw). Then, 50 μL of the mixtures were mixed with 150 μL of200 mM sodium phosphate, 50 mM phenylalanine pH 7.0. Each reaction wasmixed briefly, and the activity was determined by tracking theabsorbance at 290 nm over time (every 12-20 s over 5-20 min) using aSpectraMax® Plus³⁸⁴ or a SpectraMax® 190 (Molecular Devices) absorbancemicroplate reader. The results are shown in FIG. 7. As shown in thisFigure, additional sodium taurocholate increases the susceptibility ofVariant No. 36 to proteolysis.

Example 6 Intestinal Stability of Variant PAL

To assess the stability and activity of PAL variants as they transitthrough an animal gut, mice were gavaged with purified enzyme variants.Healthy C57Bl/6 mice, 10-12 weeks old and weighing 20-26 g, weremaintained in a metabolic cage and fasted for 15 h. Water was providedad libitum. Following the overnight fast, animals were gavaged using a21-gauge gavage needle with a mixture of 0.3 ml of 0.5 M Tris-HCl pH8.5, and 8 mg/ml in 0.5 M Tris-HCl pH 8.5, WT PAL (prepared as describedin Example 4) or 8 mg/ml in 0.5 M Tris-HCl pH 8.5 Variant No. 42(prepared as described in Example 4). At 0.5, 2, or 6 h post-gavage, theanimals were decapitated, plasma was collected using green-top capillaryblood collection tubes (Ram Scientific), and the contents of thestomach, duodenum (˜1-8 cm from the stomach), jejunum (˜10-18 cm fromthe stomach), ileum (˜8 cm above the cecum), and colon (˜5 cm below thececum) were collected. The weight of these contents was recorded and thecontents were stored at −80° C. prior to analysis.

Stomach or intestinal contents were diluted 4× with 100 mM sodiumphosphate pH 7.0, mixed briefly, and centrifuged at 14,000 rpm×2 min.The supernatants were transferred to a 350 μL, 0.45 μM, AcroPrep™Advanced 96-well filter plate (Pall Corp), and particulates were removedvia vacuum filtration. The clarified filtrate was assessed for enzymaticactivity as described in the previous Examples and for the presence ofintact PAL protein by SDS-PAGE. The results indicated that enzymaticactivity in the duodenum and jejunum appeared to be higher for theevolved PAL variants, as compared to the wild-type PAL enzyme andnegative control.

Example 7 Plasma Phenylalanine Levels

Plasma samples collected from the mice described in Example 6 wereevaluated to determine the quantity of phenylalanine present in thebloodstreams of the tested mice. Mouse plasma (50 μL) was combined with250 μL of acetonitrile containing 0.6 mM of dl-phenylalanine (Ring D₅)(i.e., an isotopically labeled version of phenylalanine comprisingdeuterium rather than hydrogen bonded with the aromatic ring carbons;Cambridge Isotope Laboratories). The samples were mixed at RT for 5 min,centrifuged at 3200×g for 10 min at 4° C. and the supernatants weretransferred to a plate for sample analysis. For the analysis, 10 μL ofeach sample was injected into a3200 QTRAP® LC/MS/MS system (AB Sciex)across a DISCOVERY® C18 column (150×2.1 mM, 5 μm beads) (Supelco, nowSigma-Aldrich), eluting with 0.1% formic acid in water (A) andacetonitrile (B). Samples were eluted across a 5 min gradient (t=0, 97%A; 3 min, 50% A; 3.5 min, 5% A; 4 min, 97% A; 5 min, 95% A) looking fora transition of 166 to 120 for endogenous phenylalanine and 171 to 125for the isotopically labeled standard. The results indicated that theplasma phenylalanine levels were lower at the 30 minute time-point insamples from mice that were given the evolved PAL variant (i.e., VariantNo. 42), as compared to the wild-type PAL enzyme and negative control.

Example 8 Therapeutic Function of Variant PAL

To assess whether PAL variants reduce serum Phe levels in vivo, a mousemodel of PKU was used. In these experiments, the PAL proteins weregavaged into affected animals. First, a consistent baseline Phe levelwas established in the mice by removing Phe from their diets for threedays followed by injection of known quantity of Phe-containing solution.Three- to six-month old homozygous PAH enu-2 mice with a C57Bl/6background (See, McDonald et al., Proc. Natl. Acad. Sci. USA87:1965-1967 [1990]) were transferred to a phenylalanine-free diet(TD.97152, Harlan) with 0.03 g/L of Phe provided in their drinking waterfor 72 h. Prior to initiating treatment at time=0 h, mice were injectedwith 0.15 mg/g of body weight Phe (from a 10 g/L solution of Phe inwater). Fifty-five minutes post-injection, approximately 20 μL of bloodwas collected by tail-vein puncture and spotted onto filter paper.Subsequently, at times 1 h-, 3 h-, and 5 h-post injection the mice weregavaged with 0.3 ml of 50-100 g/L of WT AvPAL, WT AvPAL plus aprotinin,BSA, or Variant No. 42. At 6 h-, 7 h-, and 9 h-post injection,additional blood spots were collected on filter paper. The blood spotswere dried and stored at −20° C. prior to LC-MS/MS analysis for Phe andTyr levels using methods known in the art (See, Chase et al., Clin.Chem., 39:66-71 [1993]).

The results are shown in FIG. 8. As indicated in this Figure, gavagewith inactive protein (BSA), led to increased serum Phe levels. Incontrast, treatment with proteolytically susceptible WT AvPAL resultedin constant Phe-levels, while the same protein, combined with theprotease inhibitor aprotinin resulted in a significant drop inPhe-levels. The results also show that administering the engineered-PALVariant No. 42 resulted in decreased serum Phe levels in the absence ofprotease inhibitors.

Example 9 Deimmunization of PAL

In this Example, experiments conducted to identify diversity that wouldremove T-cell epitopes from PAL are described.

Identification of Deimmunizing Diversity:

To identify amino acids that, when mutated, could remove T-cellepitopes, computational methods were used to identify PAL sequencespredicted to elicit a T-cell response. In parallel, experimentalsearches for allowable, non-deleterious mutations were also conducted,particularly for amino acids that maintain protein activity in anunchallenged assay (e.g., in the assays described in Example 2). Activevariants were then analyzed for the effect of the mutations on thepredicted immunogenicity.

Computational Identification of Putative T-cell Epitopes in a VariantAvPAL:

Putative T-cell epitopes in a AvPAL Variant No. 36 were identified usingthe Immune Epitope Database (IEDB; Immune Epitope Database and AnalysisResource website) tools, as known in the art and proprietary statisticalanalysis tools (See e.g., iedb.org and Vita et al., Nucl. Acids Res., 38(Database issue):D854-62 [2010]. Epub 2009 Nov. 11]). The AvPAL variantwas parsed into all possible 15-mer analysis frames, with each frameoverlapping the last by 14 amino acids. The 15-mer analysis frames wereevaluated for immunogenic potential by scoring their 9-mer core regionsfor predicted binding to eight common Class II HLA-DR alleles(DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101,DRB1*1301, and DRB1*1501) that collectively cover nearly 95% of thehuman population (See e.g., Southwood et al., J. Immunol., 160:3363-3373[1998]), using methods recommended on the IEDB website. Potential T-cellepitope clusters contained within the variant AvPAL (i.e., sub-regionscontained within the variant AvPAL which have an unusually highpotential for immunogenicity) were identified using statistical analysistools, as known in the art. The identified T-cell epitope clusters werescreened against the IEDB database of known epitopes and the GenBankprotein database. These screens identified 10 (ten) putative T-cellepitopes in the variant AvPAL Variant No. 36. These epitopes arereferred to as TCE-I, II, III, IV, V, VI, VII, VIII, IX, and X, below.

Design of Deimmunizing Libraries:

First, a combinatorial library containing all neutral, and beneficialmutations identified from the rounds of directed evolution used tocreate variant AvPALs, at the 10 putative T-cell epitope regionsidentified as described above was developed. The effects of thesemutations on the predicted binding to the eight common Class II HLA-DRalleles were analyzed. Multiple mutations were predicted to remove orreduce TCE-I, II, VI, VII. These mutations were combined into acombinatorial library. Libraries were then designed using saturationmutagenesis to mutagenize every single amino acid within the remainingsix T-cell epitopes (i.e., TCE-III, IV, V, VIII, IX, and X). Finally acombinatorial library was created containing beneficial diversityidentified from multiple rounds of evolution that targeted TCE-I, III,IV, VIII, and X, along with C503 and C565, two amino acids reported toimpact the aggregation state of PAL variants. The best hits from thislibrary were subjected to further saturation mutagenesis targetingTCE-III and VIII and additional targeted mutagenesis at a few positions.

Construction and Screening of Deimmunizing Libraries:

Combinatorial and saturation mutagenesis libraries designed as describedabove were constructed by methods known in the art, and tested foractivity in an unchallenged assay as described in Example 2. Activevariants were identified and sequenced. Their activities and mutationswith respect to AvPAL Variant No. 36 and numerous AvPAL Variants areprovided in Tables 9-1 through 9-7, below.

Identification of Deimmunizing Diversity:

Active variants were analyzed for their levels of immunogenicity byevaluating their binding to the eight common Class II HLA-DR allelesdescribed above. The total immunogenicity score and immunogenic hitcount are shown in Tables 9-1 to 9-7. The total immunogenicity scorereflects the overall predicted immunogenicity of the variant (i.e., ahigher score indicates a higher level of predicted immunogenicity). Theimmunogenic “hit count” indicates the number of 15-mer analysis frameswith an unusually high potential for immunogenicity (i.e., a higher hitcount indicates a higher potential for immunogenicity). Mutations with alower total predicted immunogenicity score and/or an immunogenic hitcount less than that of the reference variant were considered to be“deimmunizing mutations.” The deimmunizing mutations that wereidentified as being the best were recombined to generate a number ofvariants that are active and predicted to be significantly lessimmunogenic than the starting reference variant AvPAL. In the followingTables, the FIOP results are from the unchallenged assay; for the totalimmunogenicity score (TIS) and immunogenic hit count (IHC), the resultsare indicated for the whole PAL protein (Tables 9-1, 9-8, and 9-9) orfor the indicated epitope (Tables 9-2 to 9-7).

TABLE 9-1 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-I, II, VI, and VII Variant ActiveMutations # (as Compared to Variant No. 36) FIOP TIS IHC Variant + 68052 36 100 I27E/L214E + 634 51 101 V105C/R134Q/Q205T/P266H/L278D + 645 48102 I27E/L214E/C503Q/A547D + 629 51 103V105C/Q205T/P210C/L214E/C503Q/A547D + 619 49 104I27E/A112C/R134Q/Q205T/I285E/C503Q + 622 45 105 V39A/P266H + 665 45 106I27E/V39A/V105C/R134Q/P210C + 607 41 107I27E/V39A/V105C/P210C/L214E/P266H/L278D + 590 42 108V39A/V105C/L214E/P266H/A547D/C565N + 613 42 109 I27E/V39A/A112C/L214E +616 42 110 I27E/V39A/R134Q/A153G/L214E/P266H/I285E/C503Q/A551D/C565N +572 37 111 I27E/R134Q/L278D/I285E/A551D/C565N + 626 48 112I27E/V39A/V105C/A112C/Q205T/P266H/I285E/C503Q/A551D + 594 39 113I27E/V105C/L214E/P266H/C503Q + 614 49 114 I27E/V39A/R134Q + 637 44 115C503Q/A547D + 675 52 116 V105C/C503Q + 660 50 117I27E/R134Q/Q205T/P266H/L278D/A547D + 641 50 118I27E/V105C/R134Q/P210C/P266H/L278D/I285E/C503Q/A551D/C565N + 596 45 119I27E/V39A/V105C/R134Q/Q205T/L278D/I285E/C503Q/A547D/A551D/C565N + 585 38120 V105C/R134Q/L214E/C503Q/A547D/A551D + 619 48 121I27E/V39A/V105C/L214E/L278D/L309P/C503Q/A547D/A551D + 587 42 122V105C/L278D/C503Q/A551D + 655 50 123I27E/V105C/R134Q/P210C/L214E/P266H/L278D/A551D/C565N ++ 591 48 124I27E/R134Q/L214E/C503Q/A547D ++ 620 50 125R134Q/P210C/L214E/L278D/C503Q/A547D/C565N + 630 50 126I27E/V39A/V105C/Q205T/P210C/L214E/L278D/A547D + 585 42 127V39A/Q205T/L278D/A547D/A551D + 654 44 128V105C/R134Q/L214E/P266H/I285E/C503Q/A551D/C565N + 598 45 129I27E/V105C/L214E/A547D/A551D/C565N + 609 49 130I27E/V39A/V105C/Q205T/L278D/C503Q/A547D + 615 42 131I27E/V39A/V105C/R134Q/L214E/I285E/C503Q/A547D/A551D + 564 38 132C503Q/A547D/A551D/C565N + 675 52 133 V105C/R134Q/L214E/C503Q/A547D + 61948 134 I27E/V105C/Q205T/C503Q/A547D/A551D/C565N + 630 49 135 I27E/V39A +646 45 136 I27E/V39A/V105C/L214E/P266H/C503Q/A547D/C565N + 594 42 137A112C/R134Q/L214E/C503Q/A547D/A551D/C565N + 636 48 138I27E/A112C/R134Q/A153R/L214E/P266H/C503Q + 619 47 139 C503Q/A551D + 67552 140 I27E/V39A/V105C/R134Q/L278D/C503Q/C565N + 617 42 141R134Q/Q205T/L214E/I285E/C503Q/A551D/C565N + 613 47 142V105C/L214E/I285E/A547D/C565N + 607 46 143 I27E/V39A/R43L/L214E/A547D +625 45 144 I27E/V39A/P210C/L214E/I285E/C503Q/A551D + 584 41 145I27E/R134Q/L214E/C503Q/A547D/A551D + 620 50 146V39A/V105C/A153R/P266H/A547D/A551D + 637 42 147 V39A/C503Q + 665 45 148I27E/V39A/V105C/P210C/I285E/C503Q/A547D/A551D/C565N + 590 39 149R134Q/L214E/L278D/C503Q/A551D + 639 50 150 I27E/V39A/R134Q/C503Q/A547D +632 44 151 A153R + 677 51 152I27E/V39A/V105C/A112C/R134Q/L214E/L278D/C503Q/A547D/C565N + 585 41 153C503Q/A547D/C565N + 675 52 154I27E/V39A/L214E/P266H/L278D/C503Q/A547D/A551D/C565N + 614 44 155L278D/A547D + 675 52 156 V39A/G45S/L278D/C503Q/A551D + 667 45 157V39A/A153R/C503Q/A547D + 657 44 158R134Q/P210C/L214E/C503Q/A547D/A551D + 630 50 159 I27E/A547D/C565N + 65652 160 V39A/R134Q/P210C/L214E/A547D/C565N + 615 43 161I27E/V39A/P210C/P266H/I285E/C503Q/A547D + 610 41 162I27E/V39A/R134Q/L278D/C503Q/A547D + 632 44 163I27E/V105C/R134Q/Q205T/P210C/C503Q + 622 48 164I27E/V105C/R134Q/L214E/L278D/C503Q/A547D + 600 48 165I27E/V105C/C503Q/A547D/C565N + 636 50 166 I27E/L214E/C503Q + 634 51 167V105C/L214E/L278D/C503Q/A547D/A551D + 628 49 168I27E/V105C/Q205T/L214E/P266H + 609 49 169V39A/A112C/R134Q/L214E/C503Q/A547D/A551D + 621 41 170I27E/R134Q/L214E/C503Q/C565N + 625 50 171I27E/V39A/R134Q/A153R/Q205T/L214E/P266H/C503Q + 602 42 172I27E/V39A/L214E/C503Q/A551D + 614 44 173 V39A/V105C/Q205T/A551D + 634 42174 I27E/V39A/Q205T/C503Q/A547D/C565N + 635 44 175 A547D + 675 52 176I27E/V39A/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N + 576 41 177V39A/P275R/L278D/C503Q/A551D + 667 45 178I27E/V39A/V105C/Q205T/P210C/L278D/A547D + 611 42 179V105C/A153R/Q205T/L214E/P266H/C503Q/A547D + 620 48 180V105C/A112C/R134Q/Q205T/L214E/Y492H/C503Q/A547D + 614 48 181I27E/P210C/L278D/C503Q + 651 51 182 I27E/P210C/C503Q + 651 51 183I27E/V39A/R134Q/A153R/L214E/C503Q/A547D + 602 42 184I27E/P266H/A547D/A551D + 656 52 185V105C/L214E/I285E/C503Q/A547D/A551D/C565N + 607 46 186I27E/V105C/P266H/I285E/C503Q/A547D/C565N + 615 47 187Q205T/L278D/I285E/A547D/A551D + 648 48 188 V39A/V105C/Q205T/C503Q + 63942 189 I27E/V39A/Q205T/P266H/I285E/A547D/A551D/C565N + 614 41 190V105C/L214E/I285E + 612 46 191 V105C/R134Q/C503Q/A547D/C565N + 646 49192 I27E/V39A/V105C/R134Q/C503Q/A551D + 612 42 193I27E/R134Q/Q205T/I285E/C503Q/A551D + 620 47 194I27E/V39A/A112C/Q205T/L278D/I285E + 616 39 195I27E/V39A/A112C/Q205T/L214E/P266H/C503Q/A551D/C565N + 606 42 196I27E/V39A/L278D/A547D + 641 45 197I27E/V39A/V105C/R134Q/Q205T/L214E/A551D/C565N + 580 41 198I27E/G45D/Q205T/P266H/C565N + 656 51 199I27E/V39A/A112C/L214E/L278D/C503Q/A547D/A551D + 611 42 200 I27E/Q205T +655 51 201 I27E/A112C/R134Q/Q205T/L278D/C503Q + 643 48 202 C565N + 68052 203 I27E/V39A/V105C/R134Q/Q205T/P210C/L278D/C503Q/A547D + 602 41 204V105C/L214E/P266H/L278D/A547D + 628 49 205V105C/R134Q/A153R/Q205T/L214E/C503Q + 616 47 206 V105C/R134Q/C503Q + 65149 207 I27E/V39A/Q205T/L278D/C503Q/C565N + 640 44 208I27E/V39A/V105C/S131N/R134Q/Q205T/L214E/C503Q/A547D/C565N + 579 41 209Q205T/L214E/I285E/C503Q/A551D + 622 48 210 I27E/L214E/L278D/C503Q + 63451 211 V105C/R134Q/L214E/L278D/C565N + 624 48 212 I27E/V39A/L214E + 61944 213 L214E/C503Q/A547D + 648 51 214 L214E/P266H + 653 51 215I27E/V39A/C503Q + 646 45 216 P210C/L214E + 644 51 217 V105C + 660 50 218A112C/R134Q/A153R/L214E/L278D/I285E/C503Q/A547D/A551D/C565N + 612 44 219I27E/Q205T/L278D/A551D + 650 51 220 I27E/L214E/A551D + 629 51 221R134Q/L214E/I285E/C503Q ++ 623 47 222 I27E/V39A/V105C/C503Q/A551D + 62143 223 A112C/L278D/C503Q/A547D + 672 50 224I27E/R134Q/A153R/I285E/C503Q/A547D ++ 623 47 225I27E/V39A/V105C/A153R/I285E + 602 39 226I27E/V39A/R134Q/L278D/I285E/C503Q/A547D/A551D ++ 611 41 227R134Q/C503Q + 671 51 228 V39A/C503Q/A551D/C565N + 660 45 229I27E/V39A/P266H + 646 45 230 V105C/I285E + 639 47 231I27E/V39A/V105C/A153R/L214E + 596 41 232 A112C/L214E/L278D + 650 49 233I27E/R134Q/P210C + 642 50 234 I27E/A153R/L214E/L278D/A551D + 626 50 235V105C/I285E/A547D + 634 47 236I27E/Q205T/L214E/L278D/I285E/C503Q/C565N + 608 48 237I27E/V39A/P210C/T212S + 636 44 238 I27E/P266H/L278D/C503Q + 661 52 239I27E/V105C/Q205T/L214E/P266H/A551D/C565N + 604 49 240I27E/A112C/P210C/L214E/C503Q/A547D + 617 49 241I27E/V39A/R134Q/P210C/C503Q/A551D + 622 43 242I27E/V39A/V105C/I285E/A547D + 600 40 243I27E/V39A/Q205T/L278D/C503Q/A551D/C565N + 635 44 244I27E/R134Q/I285E/C503Q + 631 48 245I27E/V39A/P210C/L214E/L278D/C503Q/A551D + 605 44 246V39A/A112C/R134Q/Q205T/L214E/L278D + 621 41 247I27E/V39A/L214E/L278D/C503Q + 619 44 248 I27E/V105C/L214E + 614 49 249I27E/V39A/V105C/R134Q/P266H/C503Q/A547D/A551D + 612 42 250I27E/V39A/P210C/P266H/C503Q/A551D + 631 44 251I27E/V39A/V105C/A153R/C503Q/A547D/C565N + 618 42 252V39A/V105C/R134Q/L214E/C503Q/A547D/A551D + 604 41 253 I27E/L214E/L278D +634 51 254I27E/V39A/V105C/R134Q/A153R/P210C/L278D/I285E/C503Q/A547D/A551D + 578 37255 V105C/R134Q/Q205T/L214E/A547D + 614 48 256I27E/V105C/R134Q/A153R/C503Q + 629 48 257 V105C/R134Q/C503Q/A547D + 64649 258 I27E/V39A/L278D/I285E/C503Q/A547D + 620 42 259I27E/V39A/R134Q/A153R/L278D + 634 43 260I27E/V39A/A112C/R134Q/L214E/C503Q/A547D + 602 41 261I27E/V39A/R134Q/A153R/L278D/A547D/A551D + 629 43 262I27E/V39A/L278D/C503Q/C565N + 646 45 263 Q205T/L214E/C503Q/A547D/C565N +643 51 264 R134Q/L214E + 644 50 265 I27E/V39A/P266H/L278D + 646 45 266R134Q/Q205T/C503Q + 665 50 267 V39A/V105C/R134Q/P210C/L214E/A551D + 59541 268 V105C/A153R/Q205T/P266H/I285E/A547D/C565N + 625 45 269I27E/V39A/R134Q/L214E/P266H/A551D + 605 43 270L214E/P266H/C503Q/A547D/A551D/C565N + 648 51 271V39A/V105C/P210C/A547D + 630 42 272 I27E/V105C + 641 50 273I27E/V39A/R134Q/Q205T/L214E/P266H/I285E/C503Q/A551D/C565N + 579 40 274I27E/V39A/R134Q/Q205T/P266H/C503Q/A551D/C565N + 626 43 275I27E/V105C/R134Q/P210C/L214E/C503Q/A551D/C565N + 591 48 276I27E/L214E/C503Q/A547D/C565N + 629 51 277I27E/V39A/R134Q/A153R/P210/L214E/L278D/I285E/A547D/C565N + 572 39 278I27E/V39A/A112C/I285E + 622 40 279I27E/A112C/R134Q/L278D/I285E/C503Q/A551D/C565N + 623 46 280I27E/L214E/P266H/L278D/I285E/A551D + 608 48 281I27E/V39A/V105C/A153R/A551D/C565N + 618 42 282V39A/V105C/R134Q/A153R/Q205T/A551D + 622 40 283I27E/V39A/V105C/R134Q/Q205T/L214E + 585 41 284 I27E/V105C/Q205T + 635 49285 I27E/Q205T/P266H/L278D/I285E/A551D/C565N + 629 48 286I27E/R134Q/L214E/C503Q + 625 50 287V105C/Q205T/L214E/C503Q/A547D/A551D/C565N + 623 49 288V39A/V105C/L214E/I285E/C503Q/A551D/C565N + 592 39 289I27E/V39A/V105C/L278D/C503Q/A547D/C565N + 621 43 290I27E/V39A/V105C/R134Q/I285E/A547D/A551D + 591 39 291I27E/V39A/A112C/R134Q/L214E/P266H/A551D + 602 41 292I27E/L278D/C503Q/A551D + 656 52 293 I27E/V39A/R134Q/A153R/A547D + 629 43294 P266H/C565N + 680 52 295 I27E/V105C/I285E/C503Q/A547D/A551D/C565N +615 47 296 I27E/V39A/L278D + 646 45 297 P210C/L214E/P266H + 644 51 298I27E/V39A/R134Q/P210C/L214E/C503Q + 601 43 299I27E/V39A/R43L/V105C/A153R/L214E/P266H/L278D/C503Q + 607 42 300I27E/V105C/Q205T/L214E/L278D/I285E/C503Q/A547D/A551D/C565N + 583 46 301I27E/V39A/V105C/C503Q/A547D/A551D/C565N + 621 43 302I27E/V105C/L214E/I285E/A551D/C565N + 588 46 303 I27E/P210C/A551D + 64651 304 I27E/V39A/V105C/Q205T/L214E/L278D/C503Q/A547D + 589 42 305I27E/V105C/L278D/A547D + 636 50 306 C503Q + 680 52 307I27E/V39A/V105C/R134Q/L214E/L278D/A547D/A551D + 585 41 308 I27E/R134Q +652 51 309 V39A/R134Q + 656 44 310I27E/V39A/V105C/A112C/Q205T/P210C/P266H/C503Q/A547D + 611 42 311V39A/A112C/A153R/Q205T/L278D/C503Q/A547D + 648 41 312 I27E/V105C/P266H +641 50 313 I27E/V39A/V105C/R134Q/P210C/L214E + 581 41 314I27E/V39A/V105C/R134Q/L278D/A551D + 612 42 315I27E/V39A/V105C/A112C/R134Q/A153R/Q205T/L214E/P266H/L278D/C503Q/A551D +577 40 316 I27E/V105C/R134Q/A153R/I285E/A547D + 603 45 317I27E/V39A/A112C/A547D + 638 43 318I27E/V39A/R134Q/A153R/L214E/P266H/L278D/C503Q/A547D/C565N + 602 42 319I27E/V105C/Q205T/P266H/C503Q + 635 49 320I27E/V39A/V105C/R134Q/L278D/I285E/C503Q + 596 39 321 C503Q/A551D/C565N +675 52 322 I27E/V39A/A112C/R134Q/Q205T/P210C/L214E/A551D/C565N + 593 41323 R134Q/C503Q/A547D/A551D + 666 51 324I27E/A112C/Q205T/P266H/L278D/I285E/C503Q + 631 46 325P266H/L278D/C503Q + 680 52 326 I27E/V39A/Q205T/P266H/A551D + 635 44 327Q205T/L214E/I285E/C503Q/C565N + 627 48 328I27E/V105C/R134Q/Q205T/L214E/P266H/L278D/C503Q/C565N + 600 48 329I27E/V39A/V105C/R134Q/P266H/C503Q + 617 42 330I27E/V39A/V105C/R134Q/P210C/L214E/I285E/A547D + 555 38 331I27E/V39A/V105C/Q205T/P210C/L278D/C503Q + 616 42 332I27E/V39A/R134Q/A547D/C565N + 632 44 333 V105C/A547D/A551D + 655 50 334I27E/V105C/R134Q/A153R/P210C/L214E/C503Q/A547D + 588 47 335I27E/P210C/L214E/C503Q/A547D ++ 620 51 336I27E/V105C/C503Q/A547D/A551D/C565N + 636 50 337I27E/V105C/R134Q/L214E/L278D/C503Q/A547D/A551D/C565N + 600 48 338I27E/V39A/V105C/A153R/Q205T/L278D/C503Q/A547D/A551D + 612 41 339I27E/V39A/V105C/A112C/L214E/I285E/C503Q/A547D + 573 39 340 L214E/A547D +648 51 341 V105C/Q205T/L214E/L278D + 628 49 342I27E/V39A/Q205T/L214E/C503Q/C565N + 614 44 343 I27E/P210C + 651 51 344V105C/Q205T/C503Q/A551D + 649 49 345 I27E/V39A/A112C/R134Q/P266H/I285E +613 39 346 V39A/P210C/L214E/L278D/I285E/C503Q/A551D + 603 41 347I27E/Q205T/L214E/C503Q/A547D/C565N + 624 51 348I27E/V39A/L214E/L278D/C503Q/A547D/C565N + 614 44 349I27E/P210C/C503Q/C565N + 651 51 350 I27E/L278D/C503Q/A551D/C565N + 65652 351 R134Q/L214E/L278D/C503Q + 644 50 352I27E/A153R/L214E/L278D/I285E/A551D/C565N + 605 47 353I27E/V105C/L214E/A551D/C565N + 609 49 354V39A/R134Q/Q205T/L214E/C503Q/C565N + 624 43 (“−” = FIOP < 0.7; “+” =FIOP 0.7 < FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-2 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-III Variant Active Mutations # (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 71 7 36 400V80I/R134C/P564Q + 60 5 401 V121C + 44 6 402 R134L + 78 7 403 A124G + 587 404 R134W + 63 5 405 L126M + 56 5 406 N130C/M370I + 62 6 407 N130Q +65 7 408 A123G + 67 8 409 A129G + 64 9 410 G135A/A394E + 76 7 411A129L + 82 9 412 M133R + 52 3 413 G135S + 78 9 414 L126T + 62 8 415L127A + 46 6 416 R134I + 82 7 417 R134N/G307C + 60 5 418 L126I ++ 80 12419 G135C + 67 6 420 M125L + 72 8 (“−” = FIOP < 0.7; “+” = FIOP 0.7 <FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-3 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-1V Variant Active Mutations # (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 74 11 36 500 P157H +69 8 501 R140M + 72 11 502 P157F + 80 12 503 A153C + 56 7 504 E142V + 8414 505 K145G/P157T + 62 8 506 R140D + 63 8 507 E142D/G371D + 79 13 508M147A + 48 1 509 T156K/G483C + 83 12 510 P157D + 64 8 511 A62S/M147V +62 8 512 R146L + 81 14 513 Y158E + 72 11 514G154Y/L174M/Q321K/S456I/G483C + 75 10 515 S821/G135C/P157F/W279L + 77 11516 I144N + 61 7 517 T110I/I139R + 65 8 518 L47M/I144L + 67 8 519L47M/M147G/A383E + 48 4 520 G20S/I144L + 67 8 521 R146W/D191Y + 62 5 522P157Y + 73 9 523 L47M/P157C + 68 8 524 E142P + 68 9 525 F150K + 59 6 526L141T + 55 7 527 V159H + 67 8 528 I144L + 67 8 529 A119E/T156H/A289D +78 11 530 Q58K/P157D/G369C + 64 8 531 L47M/R146E + 58 6 532 E142H + 7913 533 R140N/A199E + 67 8 534 I144V + 72 13 535 I149L + 69 9 536 R146H +77 12 537 I139M + 74 11 538 A153S/H250N + 73 10 539 L319M + 74 11 540R140E/A334S/A551D + 63 8 541 F150L + 79 12 542 L143M + 72 11 543 A153G +62 8 544 I139V + 74 11 545 Q58H/L143V + 72 11 546 G154R + 81 13 547K145Q + 72 11 548 L143F + 74 11 549 R140G + 67 8 550 V159C + 72 11 551Q389K + 74 11 552 L141P + 58 5 553 M247I + 74 11 554 F150M + 77 12 555L141Q + 59 7 556 L151M + 68 9 557 V159L + 74 11 558 R94C/I149E + 53 6559 V159M + 74 11 560 L118M/L141H + 58 7 561 K145N + 71 11 562 I149R +72 11 563 K145R + 84 15 564 L141K + 59 7 565 R43S + 74 11 (“−” = FIOP <0.7; “+” = FIOP 0.7 < FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-4 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-V. Variant Active Mutations No. (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 36 2 36 600 Y176R +51 4 601 S180C + 27 0 602 S180T + 27 0 603 V172L + 30 1 604 I177V + 24 0605 V172I + 29 1 606 I177M + 30 1 607 Y176I + 54 4 608 Y176M + 33 2 609Y176V + 41 3 610 L174M + 21 0 611 P117T/Y176Q + 37 2 612 S175G + 26 0613 T178L/A477S + 43 5 614 V172C + 26 0 615 Y176E + 20 0 (“−” = FIOP <0.7; “+” = FIOP 0.7 < FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-5 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-VIII. Variant Active Mutations # (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 83 10 36 700 A232S +83 10 701 A112S/M370A/A507E + 71 10 702 Q240K/H374R + 83 10 703 S461G +83 10 704 H374D + 83 10 705 M372A + 74 10 706 L349M + 83 10 707 Y378S +74 7 708 G371H + 91 11 709 Y377N + 82 10 710 I379N + 80 8 711D191Y/H385N + 81 10 712 I379L + 83 10 713 R43S/H374R + 83 10 714Q355K/H374S + 83 10 715 P275T/H374R + 83 10 716 H374Q/P396Q + 99 14 717H385N + 81 10 718 Y378L + 78 9 719 I379C + 70 7 720 M370G + 64 10 721M372V + 76 10 722 K384R + 87 10 723 A383V + 88 13 724 M147I/H374S + 8310 725 Y378F/P404Q + 85 10 726 H374S + 83 10 727 Y378E + 55 2 728H374R/G417C + 83 10 729 L418M + 83 10 730 S525L + 83 10 731 Y378D + 60 4732 A383S + 88 10 733 D387S + 96 11 734 L382H + 78 10 735 L382C + 76 8736 G371Q + 84 10 737 H374L + 85 11 738 Y378C + 58 5 739 H374A + 83 10740 L375M + 77 10 741 H385C + 74 8 742 A334S/H374V + 85 11 743 H374R +83 10 744 H385M/P403H + 83 11 745 Y378I + 83 10 746 Y377I + 86 10 747H385G + 74 7 748 H385S/P403H + 79 9 749 H374N + 83 10 750 S187R/L381V +78 7 751 L382S + 77 9 752 Y377C + 75 7 753 L381V + 78 7 754 G371S + 8810 755 A256S/L381N + 70 6 756 I379M + 80 10 757 R43S/H374K + 83 10 758I379H + 76 7 759 M370S + 64 10 760 P275Q/M370S + 64 10 761 G425V + 83 10762 A447S + 83 10 763 L382M + 80 10 764 G371N + 84 10 765 L381M/Q560K +79 10 767 L382I + 77 8 768 H374G + 83 10 769 M370I + 76 10 770Q332K/Y377M + 86 10 771 Y378N + 64 5 772 L375I + 83 10 773 H374T + 83 10774 L381G + 60 4 (“−” = FIOP < 0.7; “+” = FIOP 0.7 < FIOP < 1.4; “++” =FIOP > 1.4)

TABLE 9-6 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-IX. Variant Active Mutations No. (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 45 4 36 800 I439V +49 4 801 I439C + 39 1 802 S438F + 50 4 803 T433S + 44 0 804 T433A + 35 0805 Y435Q/H446N + 43 2 806 L431V + 28 0 807 N437Q + 40 0 808L213M/S438L + 46 4 809 L432C + 39 2 810 L432V + 47 4 811 T433L + 50 4812 L431P + 18 0 813 I439F + 47 4 814 S286R/Y435T + 53 4 815 S98I + 45 4816 S438R + 48 4 817 S331I + 45 4 818 S438M + 44 4 819 Q240K/T433Y + 410 820 T433I + 46 3 821 T433N + 38 0 822 L431S + 27 0 823 N437G + 38 0824 L431E + 26 0 825 L431C + 25 0 826 G436T + 45 0 827 G436D + 42 1 828T433V + 59 7 829 N437T/L538M + 52 4 830 A289S/L431E + 26 0 831 T433Q +37 0 832 I439L + 46 4 833 F434C + 34 0 834 T433P + 35 0 835 A24S/F434M +69 8 836 L431G + 18 0 837 S438C + 45 1 838 T433W + 33 0 839 Y435L + 57 5840 A62S/T433N + 38 0 841 N437E + 46 3 842 A477S + 45 4 843 G436M + 8711 844 T433R + 70 10 845 S438T + 46 4 (“−” = FIOP < 0.7; “+” = FIOP 0.7< FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-7 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-X. Variant Active Mutations # (asCompared to Variant No. 36) FIOPC TIS IHC Variant + 47 7 36 900 I471V +45 7 901 F472G + 16 0 902 G483S + 60 9 903 G483A/S524I + 64 10 904Y475C + 20 0 905 I478N + 44 6 906 V476I + 30 2 907 N474D/R490H + 29 3908 P275Q + 47 7 909 Q473K + 55 7 910 G483H + 58 8 911 Q473H/A507S + 527 912 N474H + 49 7 913 Y475F + 58 7 914 I478S + 46 6 915 Q473M + 69 9916 V476C + 23 0 917 L104M/V476L + 36 3 918 A119E/G365A + 47 7 919N474W + 51 7 920 I471K + 29 2 921 Q292H/A479G + 35 2 922 G276V + 47 7923 I471N + 26 1 924 A479S + 53 7 925 A558S + 47 7 926 Q473S + 56 8 927Q473R + 62 8 928 Y475Q + 33 5 929 I471G + 32 3 930 A479G + 35 2 931A70S/N474E + 32 3 932 I471F + 48 7 933 Q58R/Y475H + 32 5 934 F482L + 569 935 V476L + 36 3 936 A24E + 47 7 937 N474R + 55 7 938 G483C + 49 6 939Q473H + 52 7 940 I471M + 46 7 941 I471R + 31 2 942 P404T/A477V + 47 6943 G483R/G537C + 78 13 944 F482C + 45 6 945 Q355H/I478C + 31 1 946L206M + 47 7 947 N474A + 48 7 948 Y475L + 60 7 949 I471W + 34 4 (“−” =FIOP < 0.7; “+” = FIOP 0.7 < FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-8 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-I, III, IV, VIII, and X Variant ActiveMutations # (as Compared to Variant No. 36) FIOP TIS IHC Variant + 36950 F472G/C503Q/C565N + 651 45 951 L381G/F472G/C503Q/C565N + 628 39 952M133R/C503Q + 663 48 953 M133R/L381G/C565N + 640 42 954V39A/C503Q/C565N + 667 45 955 V39A/F472G/C503Q/C565N + 636 38 956V39A/G248C/L381G/ + 613 32 F472G/C503Q/C565N 957 V39A/K115E/M133R/C565N++ 646 39 958 V39A/L381G + 644 39 959 V39A/M133R/C503Q + 648 41 960V39A/M133R/C503Q/C565N + 648 41 961 V39A/M133R/F472G/C503Q/C565N + 61734 962 V39A/M133R/F472G/C565N + 617 34 963 V39A/M147A/C565N + 641 35 964V39A/M147A/F472G/C503Q/C565N + 610 28 965 V39A/M147A/F472G/C565N + 61028 966 V39A/M147A/L381G/C503Q/C565N + 618 29 967 V39A/M147A/L381G/ + 58722 F472G/C503Q/C565N 968 V39A/M147A/Y378E/C503Q/C565N + 613 27 969V39A/M147A/Y378E/C565N + 613 27 970 V39A/Y378E/C503Q/C565N + 639 37 971V39A/Y378E/C565N + 639 37 972 Y378D/C503Q + 659 46 973Y378E/F472G/C503Q/C565N + 623 37 (“−” = FIOP < 0.7; “+” = FIOP 0.7 <FIOP < 1.4; “++” = FIOP > 1.4)

TABLE 9-9 FIOP, Total Immunogenicity (TIS), and Immunogenic Hit Count(IHC) for Variants Targeting TCE-III and VIII Variant Active Mutations #(as Compared to Variant No. 967) FIOP TIS IHC Variant + 967 974 A129I −598 26 975 A129V − 595 24 976 A136K ++ 595 22 977 A24E/G381L + 604 28978 A289S + 590 23 979 A383C + 577 18 980 A383M ++ 602 28 981 G381A ++598 24 982 G381C − 580 22 983 G381F + 601 25 984 G381I ++ 610 27 985G381L ++ 610 28 986 G381M + 606 28 987 G381N + 597 24 988 G381Q + 590 23989 G381S + 601 24 990 G381T + 590 23 991 H132L − 603 25 992 H132S − 59223 993 H374G + 587 22 994 H374M + 589 23 995 H374Q − 587 22 996 L127V −576 22 997 L431M + 591 22 998 L563M − 587 22 999 M372L − 580 22 1000R134C − 576 20 1001 R134F + 588 21 1002 R134H + 579 21 1003 R134K − 58521 1004 S131C − 579 21 1005 S131T + 580 21 1006 V388C + 577 21 1007V388T + 578 21 1008 R134H/V388T + 570 20 1009 R134H/Y378E/G381L + 574 191010 R134H/Y378E/G381L/V388T + 566 19 (“−” = FIOP < 0.7; “+” = FIOP 0.7< FIOP < 1.4; “++” = FIOP > 1.4)In Vitro Testing of Deimmunized PAL Variants:

The deimmunized PAL variants are tested in a dendritic T-cell assay toempirically test their capacity to elicit a T-cell response. Peripheralblood mononuclear cells (PBMCs) are isolated from a human donor usingstandard techniques. These cells are used as a source of monocytes thatare cultured in defined media to generate immature dendritic cells.These immature dendritic cells (DCs) are loaded with deimmunized PALvariants, and are then induced into a more mature phenotype by furtherculturing in defined media to provide antigen-primed DCs. CD8+ Tcell-depleted donor PBMCs obtained from the same donor samples as theDCs are labeled with CFSE, then cultured with the antigen-primed DCs for7 days, after which octuplicates are tested. Each DC-T cell cultureincludes a set of untreated controls (i.e., negative controls). Theassay also incorporates reference antigen controls (i.e., positivecontrols), comprising two potent whole protein antigens. Assaysutilizing cells isolated from 50 human donors with diverse MajorHistocompatibility Complex Class II alleles provide a statisticallyrelevant assessment of the PAL variants' capacity to elicit a T-cellresponse.

While the invention has been described with reference to the specificembodiments, various changes can be made and equivalents can besubstituted to adapt to a particular situation, material, composition ofmatter, process, process step or steps, thereby achieving benefits ofthe invention without departing from the scope of what is claimed.

For all purposes in the United States of America, each and everypublication and patent document cited in this disclosure is incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute an admission as to its contents or date.

What is claimed is:
 1. An engineered polynucleotide encoding apolypeptide comprising an amino acid sequence having at least 90%sequence identity to reference sequence SEQ ID NO:4, wherein said aminoacid sequence comprises a glycine at position 290, and wherein thepositions in said amino acid sequence are in reference to SEQ ID NO:4.2. The engineered polynucleotide of claim 1, wherein said encodedengineered polypeptide comprises at least one or more of the followingsubstitutions or substitution sets selected from39/91/158/180/195/256/290/399/459/463,39/91/158/180/290/394/399/474/522/524,39/91/256/290/394/399/404/407/522/524,91/158/243/256/290/399/407/459/463/474/522/524, and256/290/404/407/474/522, wherein said positions in said amino acidsequence are in reference to SEQ ID NO:4.
 3. The engineeredpolynucleotide of claim 1, wherein said encoded engineered polypeptidecomprises at least one or more of the following substitutions orsubstitution sets selected from 39/91/256/290/307/399/404/407/522/524,39/91/256/290/307/404/407/524, 39/91/256/290/399/404/407/522,39/91/290/307/407, 39/91/290/307/407/524, and 39/256/290/307/404/407,wherein said positions in said amino acid sequence are in reference toSEQ ID NO:4.
 4. The engineered polynucleotide encoding an engineeredpolypeptide of claim 1, wherein said engineered polypeptide exhibits animproved property selected from reduced sensitivity to proteolysis,increased tolerance to acidic pH, reduced immunogenicity, or acombination thereof, as compared to the reference sequence SEQ ID NO:4.5. The engineered polynucleotide encoding an engineered polypeptide ofclaim 4, wherein the improved property is selected from reducedsensitivity to proteolysis and/or increased tolerance to acidic pH. 6.The engineered polynucleotide encoding an engineered polypeptide ofclaim 4, wherein said engineered polypeptide is resistant toproteolysis, acid stable, and/or deimmunized.
 7. The engineeredpolynucleotide encoding an engineered polypeptide of claim 5, whereinsaid engineered polypeptide is resistant to proteolysis by at least onedigestive tract enzyme, wherein said engineered polypeptide is resistantto proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/orelastases.
 8. The engineered polynucleotide encoding an engineeredpolypeptide of claim 6, wherein said engineered polypeptide isdeimmunized.
 9. The engineered polynucleotide encoding an engineeredpolypeptide of claim 1, wherein said polypeptide is purified.
 10. Theengineered polynucleotide of claim 1, wherein said polynucleotidesequence is operably linked to a control sequence.
 11. The engineeredpolynucleotide claim 1, wherein said polynucleotide sequence iscodon-optimized.
 12. An expression vector comprising at least oneengineered polynucleotide sequence of claim 1, and at least one controlsequence.
 13. An expression vector comprising at least one engineeredpolynucleotide sequence of claim 11, and at least one control sequence.14. The expression vector of claim 12, wherein said control sequence isa promoter.
 15. The expression vector of claim 13, wherein said controlsequence is a promoter.
 16. A host cell transformed with the expressionvector of claim
 12. 17. A host cell transformed with the expressionvector of claim
 13. 18. A host cell comprising the engineeredpolynucleotide of claim
 1. 19. A method of producing an engineeredpolypeptide in a host cell comprising culturing the host cell of claim16, under suitable culture conditions, such that at least one engineeredpolypeptide is produced.
 20. A method of producing an engineeredpolypeptide in a host cell comprising culturing the host cell of claim17, under suitable culture conditions, such that at least one engineeredpolypeptide is produced.
 21. A method of producing an engineeredpolypeptide in a host cell comprising culturing the host cell of claim18, under suitable culture conditions, such that at least one engineeredpolypeptide is produced.
 22. The method of claim 19, further comprisingrecovering at least one engineered polypeptide from the culture and/orhost cells.
 23. The method of claim 20, further comprising recovering atleast one engineered polypeptide from the culture and/or host cells. 24.The method of claim 21, further comprising recovering at least oneengineered polypeptide from the culture and/or host cells.
 25. Themethod of claim 22, further comprising the step of purifying said atleast one engineered polypeptide.
 26. The method of claim 23, furthercomprising the step of purifying said at least one engineeredpolypeptide.
 27. The method of claim 24, further comprising the step ofpurifying said at least one engineered polypeptide.